Activation of Na+-H+ exchange and stretch-activated channels underlies the slow inotropic response to stretch in myocytes and muscle from the rat heart

Abstract

We present the first direct comparison of the major candidates proposed to underlie the slow phase of the force increase seen following myocardial stretch: (i) the Na(+)-H(+) exchanger (NHE) (ii) nitric oxide (NO) and the ryanodine receptor (RyR) and (iii) the stretch-activated channel (SAC) in both single myocytes and multicellular muscle preparations from the rat heart. Ventricular myocytes were stretched by approximately 7% using carbon fibres. Papillary muscles were stretched from 88 to 98% of the length at which maximum tension is generated (L(max)). Inhibition of NHE with HOE 642 (5 microm) significantly reduced (P < 0.05) the magnitude of the slow force response in both muscle and myocytes. Neither inhibition of phosphatidylinositol-3-OH kinase (PtdIns-3-OH kinase) with LY294002 (10 microm) nor NO synthase with L-NAME (1 mm) reduced the slow force response in muscle or myocytes (P > 0.05), and the slow response was still present in the single myocyte when the sarcoplasmic reticulum was rigorously inhibited with 1 microm ryanodine and 1 microm thapsigargin. We saw a significant reduction (P < 0.05) in the slow force response in the presence of the SAC blocker streptomycin in both muscle (80 microm) and myocytes (40 microm). In fura 2-loaded myocytes, HOE 642 and streptomycin, but not L-NAME, ablated the stretch-induced increase in [Ca(2+)](i) transient amplitude. Our data suggest that in the rat, under our experimental conditions, there are two mechanisms that underlie the slow inotropic response to stretch: activation of NHE; and of activation of SACs. Both these mechanisms are intrinsic to the myocyte.

Figure 1. The inotropic response to stretch in a ventricular myocyte and papillary muscle from the rat

A, myocyte attached to a single supple carbon fibre (left) and a double stiff fibre (right). See Methods for further details. B, representative response to stretch in a myocyte, recorded through a sample and hold circuit to show only changes in active force. Sarcomere length was increased from 1.85 to 2.0 μm (↑) and released (↓) at 5 min. C, representative changes in [Ca²⁺]_i (expressed as fura-2 ratio units) in a myocyte following stretch from 1.81 to 1.90 μm. Traces represent an average of 10 transients recorded at short length (dark grey), between 5 and 15 s following stretch (mid grey) and at 5 min following stretch (light grey). Transients at short length and immediately following stretch are virtually superimposed. D, representative force response to stretch in a papillary muscle. Muscle length was increased from 88 to 98%L_max (↑) and released to 88%L_max at 10 min (↓). All these data were obtained in the presence of physiological bicarbonate-buffered bathing solution.

Figure 2. Stimulation of Na⁺–H⁺ exchange contributes to the slow response to stretch in both papillary muscle and ventricular myocytes from the rat

Magnitude of the slow force response in muscle (A) and myocytes (B) under control conditions (open bars), after exposure to the NHE inhibitor HOE 642 (5 μm; hatched bars), and following washout of HOE 642 (spotted bar). Myocytes were stretched by 7.9 ± 1.5% from a resting sarcomere length of 1.84 ± 0.01 μm; *P < 0.05, **P < 0.01 compared with control group (paired Student's t test) C, [Ca²⁺]_i transient amplitude at 10 s and 5 min after stretch in fura 2-loaded myocytes stretched by 5.7 ± 0.7% from a resting sarcomere length of 1.82 ± 0.01 μm; *P < 0.05 compared with control group at 10 s (paired Student's t test). All data are mean + s.e. (n = 4–6).

Figure 3. Nitric oxide signalling does not contribute to the slow inotropic response to stretch in papillary muscle or ventricular myocytes from the rat

The magnitude of the slow response to stretch under control conditions (open bars), in the presence of the NO synthase inhibitor l-NAME (1 mm; hatched bars) and following washout (spotted bar) in muscle (A) and myocytes (B). Myocytes were stretched by 8.3 ± 1.2% from a resting sarcomere length of 1.83 ± 0.01 μm. C, [Ca²⁺]_i transient amplitude at 10 s and 5 min after stretch in fura 2-loaded myocytes stretched by 7.3 ± 1.0% from a resting sarcomere length of 1.87 ± 0.01 μm; *P < 0.05, **P < 0.01 compared with respective group at 10 s (paired Student's t test). All bars are mean + s.e. (n = 6–8).

Figure 4. PtdIns-3-OH kinase does not contribute to the slow inotropic response to stretch in papillary muscle or ventricular myocytes from the rat

Magnitude of the slow response in muscle (A) and myocytes (B) under control conditions (open bars), after exposure to the PtdIns-3-OH kinase inhibitor LY294002 (10 μm; hatched bars), and following washout of LY294002 (spotted bar). Myocytes were stretched by 8.2 ± 0.01% from a resting sarcomere length of 1.81 ± 0.02 μm. All data are mean + s.e. (n = 6–8). *P < 0.05 compared with control group (paired Student's t test).

Figure 5. The slow inotropic response to stretch in rat ventricular myocytes does not require a functional sarcoplasmic reticulum

A, the response of a myocyte to stretch from a sarcomere length of 1.85 to 2.0 μm under control conditions. B, the response of the same myocyte to the same degree of stretch following 10 min exposure to 1 μm thapsigargin and 1 μm ryanodine. Traces show active force only, note the difference in force scale between A and B. C, comparison of the magnitude of the slow inotropic response to stretch under control conditions (open bar) and in the presence of 1 μm ryanodine and 1 μm thapsigargin (RY + THAPS; hatched bar). Myocytes were stretched by 6.7 ± 0.7% from a resting sarcomere length of 1.84 ± 0.01 μm. Data are mean + s.e. (n = 5 cells).

Figure 6. The stretch activated channel (SAC) contributes to the slow response to stretch in papillary muscle and ventricular myocytes from the rat

Magnitude of the slow force response in muscle (A) and myocytes (B) under control conditions (open bars), after exposure to the SAC blocker streptomycin (STREP; hatched bars), and following washout of streptomycin (spotted bar). Muscles were exposed to 80 μm streptomycin; myocytes were exposed to 40 μm streptomycin. Myocytes were stretched by 6.7 ± 0.8% from a resting sarcomere length of 1.85 ± 0.01 μm *P < 0.05 compared with control group (paired Student's t test). C, [Ca²⁺]_i transient amplitude at 10 s and 5 min after stretch in fura 2-loaded myocytes stretched by 5.7 ± 0.7% from a resting sarcomere length of 1.90 ± 0.02 μm **P < 0.01 compared with control group at 10 s (paired Student's t test). All data are mean + s.e. (n = 5–7).