Isometric Stretch Alters Vascular Reactivity of Mouse Aortic Segments

Abstract

Most vaso-reactive studies in mouse aortic segments are performed in isometric conditions and at an optimal preload, which is the preload corresponding to a maximal contraction by non-receptor or receptor-mediated stimulation. In general, this optimal preload ranges from about 1.2 to 8.0 mN/mm, which according to Laplace's law roughly correlates with transmural pressures of 10-65 mmHg. For physiologic transmural pressures around 100 mmHg, preloads of 15.0 mN/mm should be implemented. The present study aimed to compare vascular reactivity of 2 mm mouse (C57Bl6) aortic segments preloaded at optimal (8.0 mN/mm) vs. (patho) physiological (10.0-32.5 mN/mm) preload. Voltage-dependent contractions of aortic segments, induced by increasing extracellular K⁺, and contractions by α₁-adrenergic stimulation with phenylephrine (PE) were studied at these preloads in the absence and presence of L-NAME to inhibit basal release of NO from endothelial cells (EC). In the absence of basal NO release and with higher than optimal preload, contractions evoked by depolarization or PE were attenuated, whereas in the presence of basal release of NO PE-, but not depolarization-induced contractions were preload-independent. Phasic contractions by PE, as measured in the absence of external Ca²⁺, were decreased at higher than optimal preload suggestive for a lower contractile SR Ca²⁺ content at physiological preload. Further, in the presence of external Ca²⁺, contractions by Ca²⁺ influx via voltage-dependent Ca²⁺ channels were preload-independent, whereas non-selective cation channel-mediated contractions were increased. The latter contractions were very sensitive to the basal release of NO, which itself seemed to be preload-independent. Relaxation by endogenous NO (acetylcholine) of aortic segments pre-contracted with PE was preload-independent, whereas relaxation by exogenous NO (diethylamine NONOate) displayed higher sensitivity at high preload. Results indicated that stretching aortic segments to higher than optimal preload depolarizes the SMC and causes Ca²⁺ unloading of the contractile SR, making them extremely sensitive to small changes in the basal release of NO from EC as can occur in hypertension or arterial stiffening.

Keywords: aorta; contraction; isometric; nitric oxide; preload; relaxation; stretch.

Figure 1

Relationship between the estimated aortic external circumference and preload (PL). Aortic segments were clamped at preloads of 16, 30, 40, and 50 mN and aortic circumferences were determined. Data at 16, 30, and 40 mN were fitted with linear regression (line: circumference (mm) = 0.04^*PL + 2.32). The estimated diameter at 50 mN was lower than calculated (^**p < 0.01, n = 11).

Figure 2

Depolarization of aortic segments at different preloads. EC₅₀ (A) and E_max (B) values of K⁺ concentration-response curves for depolarization of aortic segments at different preloads in the absence (eNOS active, white) and presence of 300 μM L-NAME (eNOS inactive, black). The shifts of EC₅₀ and E_max caused by inhibition of eNOS with 300 μM L-NAME are shown in (C,D). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001: 30, 40, or 50 vs. 16 mN; #p < 0.05, ###p < 0.001 control vs. L-NAME, n = 7.

Figure 3

Effects of removal of external Ca²⁺ on basal force at different preloads. Decrease of basal force upon removal of extracellular Ca²⁺ in segments initially preloaded at 10, 20, 28, 40, and 65 mN (different symbol colors). (n = 5).

Figure 4

Effects of levcromakalim on K⁺ concentration-contraction curves at 16 and 40 mN. K⁺ concentration-contraction curves are shifted more to the right by levcromakalim at the preload of 40 mN (black) than at 16 mN (white). Isometric contractions were measured at elevated external K⁺ in the absence (circles) and presence (squares) of 1 μM levcromakalim and in the absence (A) and presence (B) of 300 μM L-NAME. In (C) the absolute shifts of the EC of K⁺ by levcromakalim are shown at 25, 50, and 75% contraction (EC₂₅, EC₅₀ and EC₇₅). Because shifts in the absence and presence of L-NAME did not differ, the mean shift for each segment (mouse) was determined and plotted. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; 40 vs. 16 mN, n = 4.

Figure 5

α1-Adrenoceptor stimulation of aortic segments with phenylephrine at different preloads. Log(EC₅₀) (A) and E_max (B) of PE-induced contractions at different preloads in the absence (control) and presence of L-NAME (+L-NAME, eNOS inhibited). Figures (C,D) show the shifts of EC₅₀ and of E_max by eNOS inhibition with L-NAME. ^*p < 0.05, ^**p < 0.01 ^***p < 0.001: 30, 40, or 50mN vs. 16mN ###p < 0.001 control vs. L-NAME.

Figure 6

Phasic contractions of phenylephrine at different preloads. Contractions induced by 1 μM PE in the presence of L-NAME to inhibit basal release of NO in the absence of extracellular Ca²⁺ at preloads from 10 to 65 mN. Phasic contractions as shown in (A) were fitted with a bi-exponential time course revealing amplitudes (A_on, B) and time constants (τ_on, C) contraction and time constants of relaxation (τ_off, D). ^*p < 0.05, ^**p < 0.01 vs. 20 mN, n = 5.

Figure 7

Tonic contractions of phenylephrine at different preloads. Contractions were induced at preloads from 10 to 65 mN by re-addition of extracellular Ca²⁺ to segments, which were contracted by 1 μM PE in the absence of external Ca²⁺ (see Figure 6). All contractions were in the presence of L-NAME to inhibit basal release of NO. Tonic contractions after re-addition of external Ca²⁺ as shown in (A) were at 600 s inhibited with 35 μM diltiazem. Amplitudes of the contraction at 600 s are shown in (B). In (C) the absolute amount of force decrease by diltiazem was plotted against the maximal effect of PE at the different preloads (different colors) and revealed a linear relationship with slope of 0.37. There was a highly significant (P < 0.0001) correlation (Pearson correlation r = −0.86). Hence, the relative effect of diltiazem (D) was load-independent (linear regression line deviates non-significantly from zero). Relationship between PE-induced IP₃-mediated contraction in the absence of external Ca²⁺ (see Figure 6) and the contraction elicited upon re-introduction of external Ca²⁺ (E) or the decrease of this contraction by 35 μM diltiazem (F, a measure of the VGCC-mediated component) at different preloads (10 mN: red, 20 mN: white, 28 mN: black, 40 mN: blue and 65 mN: green). There was a highly significant (P < 0.0001) correlation (Pearson correlation r = 0.79 for A and –0.86 for B) between both parameters. ^*p < 0.05 vs. 20 mN, n = 5.

Figure 8

VGCC and NSCC contribute to PE-mediated contractions at 16 and 40 mN. PE concentration-response curves for isometric contractions at preloads of 40 mN (black) or 16 mN (white) in the absence (A,C) and presence (B,D) of 300 μM L-NAME were due to Ca²⁺ influx via VGCC and NSCC. Isometric contractions in the presence of 1 μM levcromakalim reveal contractions due to Ca²⁺ influx via NSCC only (C,D). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, 40 vs. 16 mN preload (n = 4).

Figure 9

NSCC-mediated contractions are dependent on NO and preload. Relative contribution of NSCC-mediated contractions to the total contraction by PE at 16 (white) and 40 mN (black) in the absence (control) and presence of L-NAME to block basal release of NO. ^***p < 0.001, 40 vs. 16 mN, n = 4.

Figure 10

Relaxation of PE-precontracted aortic segments by endogenous and exogenous NO at different preloads. DRCs for ACh- (A) and DEANO- (B) induced relaxations at the different preloads. Pre-contractions were elicited with the EC50 concentration of PE in the absence (A) and presence (B) of 300 μM L-NAME. The log(EC50)-values of ACh (C) and DEANO (D) were calculated at the different preloads (n = 6); ^*P < 0.05, ^**P < 0.01, 16 vs. 30 mN; ^&&&P < 0.001, 16 vs. 40 mN; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, 16 vs. 50 mN.