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Background/Aims: Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na+/H+ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE-1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. Methods: Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na+-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. Results: The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. Conclusion: Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation. [ABSTRACT FROM AUTHOR]

Background The involvement of NHE-1 hyperactivity, critical for pathological cardiac hypertrophy (CH), in physiological CH has not been elucidated yet. Stimulation of NHE-1 increases intracellular Na + and Ca 2 + favouring calcineurin activation. Since myocardial stretch, an activator of NHE-1, is common to both types of CH, we speculate that NHE-1 hyperactivity may also happen in physiological CH. However, calcineurin activation is characteristic only for pathological hypertrophy. We hypothesize that an inhibitory AKT-dependent mechanism prevents NHE-1 hyperactivity in the setup of physiological CH. Methods Physiological CH was induced in rats by swimming (90 min/day, 12 weeks) or in cultured isolated cardiomyocytes with IGF-1 (10 nmol/L). Results Training induced eccentric CH development (left ventricular weight/tibial length: 22.0 ± 0.3 vs. 24.3 ± 0.7 mg/mm; myocyte cross sectional area: 100 ± 3.2 vs. 117 ± 4.1 %; sedentary (Sed) and swim-trained (Swim) respectively; p < 0.05] with decreased myocardial stiffness and collagen deposition [1.7 ± 0.05 % (Sed) vs. 1.4 ± 0.09 % (Swim); p < 0.05]. Increased phosphorylation of AKT, ERK1/2, p90 RSK and NHE-1 at the consensus site for ERK1/2-p90 RSK were detected in the hypertrophied hearts (P-AKT: 134 ± 10 vs. 100 ± 5; P-ERK1/2: 164 ± 17 vs. 100 ± 18; P-p90 RSK : 160 ± 18 vs. 100 ± 9; P-NHE-1 134 ± 10 vs. 100 ± 10; % in Swim vs. Sed respectively; p < 0.05). No significant changes were detected neither in calcineurin activation [calcineurin Aβ 100 ± 10 (Sed) vs. 96 ± 12 (Swim)], nor NFAT nuclear translocation [100 ± 3.11 (Sed) vs. 95 ± 9.81 % (Swim)] nor NHE-1 expression [100 ± 8.5 (Sed) vs. 95 ± 6.7 % (Swim)]. Interestingly, the inhibitory phosphorylation of the NHE-1 consensus site for AKT was increased in the hypertrophied myocardium (151.6 ± 19.4 (Swim) vs. 100 ± 9.5 % (Sed); p < 0.05). In isolated cardiomyocytes 24 hours IGF-1 increased cell area (114 ± 1.3 %; p < 0.05) and protein/DNA content (115 ± 3.9 %, p < 0.05), effects not abolished by NHE-1 inhibition with cariporide (114 ± 3 and 117 ± 4.4 %, respectively). IGF-1 significantly decreased NHE-1 activity during pH i recovery from sustained intracellular acidosis (J H + at pH i 6.8: 4.08 ± 0.74 and 9.09 ± 1.21 mmol/L/min, IGF-1 vs. control; p < 0.05), and abolished myocardial slow force response, the mechanical counterpart of stretch-induced NHE-1 activation. Conclusions NHE-1 hyperactivity seems not to be involved in physiological CH development, contrary to what characterizes pathological CH. We propose that AKT, through an inhibitory phosphorylation of the NHE-1, prevents its stretch-induced activation. This posttranslational modification emerges as an adaptive mechanism that avoids NHE-1 hyperactivity preserving its housekeeping functioning. [ABSTRACT FROM AUTHOR]

Background Reactive oxygen species, such as superoxide, are being increasingly recognized as key components of a vast array of signaling pathways. Angiotensin II is a well-recognized stimulus for superoxide production through NADPH oxidase activation and opening of the mitochondrial ATP-sensitive potassium channels (mK ATP ). A role for this mechanism has been proposed to explain several physiological effects of the peptide. The aim of this study was to evaluate the involvement of this mechanism in the inotropic response to 100 nmol/L angiotensin II. Methods Sarcomere shortening and intracellular pH (BCECF-epifluorescence technique) were evaluated in isolated cat ventricular myocytes placed in a perfusion chamber on the stage of an inverted microscope. Myocardial superoxide production was evaluated by the lucigenin quimioluminiscence method. Results Angiotensin II (100 nmol/L) increased~70% sarcomere shortening, effect that was only partially prevented by NADPH oxidase inhibition, mK ATP channel blockade or inhibition of the cardiac Na + /H + exchanger (NHE-1). Moreover, angiotensin II stimulates NHE-1 activity by a NADPH oxidase-dependent mechanism. Myocardial superoxide production was also increased by angiotensin II, and this action was completely prevented either by NADPH oxidase inhibition or mK ATP channel blockade. Conclusions The positive inotropic response to 100 nmol/L angiotensin II is due to both ROS/NHE-1 dependent and independent pathways, this being a point of divergence with the signaling previously described to be triggered by lower concentrations of angiotensin II (i.e.: 1 nmol/L). [ABSTRACT FROM AUTHOR]