Cardiac up-regulation of NBCe1 emerges as a beneficial consequence of voluntary wheel running in mice.

Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, µm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice. These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.

Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.

KEY MESSAGE: Oxalotrophic Stenotrophomonas isolated from tomato rhizosphere are able to protect plants against oxalate-producing pathogens by a combination of actions including induction of plant defence signalling callose deposition and the strengthening of plant cell walls and probably the degradation of oxalic acid. Oxalic acid plays a pivotal role in the virulence of the necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum. In this work, we isolated two oxalotrophic strains (OxA and OxB) belonging to the bacterial genus Stenotrophomonas from the rhizosphere of tomato plants. Both strains were capable to colonise endophytically Arabidopsis plants and protect them from the damage caused by high doses of oxalic acid. Furthermore, OxA and OxB protected Arabidopsis from S. sclerotiorum and B. cinerea infections. Bacterial inoculation induced the production of phenolic compounds and the expression of PR-1. Besides, both isolates exerted a protective effect against fungal pathogens in Arabidopsis mutants affected in the synthesis pathway of salicylic acid (sid2-2) and jasmonate perception (coi1). Callose deposition induced by OxA and OxB was required for protection against phytopathogens. Moreover, B. cinerea and S. sclerotiorum mycelial growth was reduced in culture media containing cell wall polysaccharides from leaves inoculated with each bacterial strain. These findings suggest that cell walls from Arabidopsis leaves colonised by these bacteria would be less susceptible to pathogen attack. Our results indicate that these oxalotrophic bacteria can protect plants against oxalate-producing pathogens by a combination of actions and show their potential for use as biological control agents against fungal diseases.

p38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

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 NHE1 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.

Silencing of the Na+/H+ exchanger 1(NHE-1) prevents cardiac structural and functional remodeling induced by angiotensin II.

Chronic activation of the renin angiotensin system (RAS) favors several cardiac diseases, among which myocardial hypertrophy occupies an outstanding place. In this context, the hyperactivity of the cardiac Na+/H+ (NHE-1) exchanger plays a key role. The pathologic remodeling of the myocardium constitutes an independent risk factor for morbidity and mortality with continuously increasing healthcare cost. Therefore, the development of better therapeutic strategies emerges as highly mandatory. Our goal was to prevent angiotensin II (ANGII)-induced cardiac hypertrophy by NHE-1 gene silencing in Wistar rats. The intramyocardial injection of a lentivirus coding a specific small interference RNA (l-shNHE1) significantly reduced NHE-1 expression exclusively in the heart (~ 50%) and prevented cardiac remodeling in rats exposed to chronic infusion of ANG II (heart weigh/tibia length: 24,03 ±â€¯0,7915 mg/mm vs 28,45 ±â€¯0,9779 mg/mm and collagen volume fraction 2526 ±â€¯0,5003 vs 5982 ±â€¯1043 in l-shNHE1 + ANGII and ANGII, respectively). Interestingly, this was accompanied by an improvement in cardiac function determined by echocardiography even though blood pressure remained elevated (Fractional shortening 0,5960 ±â€¯0,4228 vs -0,9567 ±â€¯0,06888 and blood pressure at the end of ANGII treatment 141,2 ±â€¯6117 mmHg vs 134,1 ±â€¯6723 mmHg; in l-shNHE1 + ANGII and ANGII, respectively). ANGII infusion increased myocardial NADPH oxidase activity but the l-shNHE1 injection prevented oxidative stress as revealed by the normalization of lipid peroxidation (T-BARS 12,40 ±â€¯2887.vs 23,05 ±â€¯1537 in l-shNHE1 + ANGII and ANGII, respectively). These results allow as to propose the partial silencing of the cardiac NHE-1 through lentiviral injection as a promising tool in the prevention of ANGII-induced cardiac hypertrophy.

Physiological cardiac hypertrophy: critical role of AKT in the prevention of NHE-1 hyperactivity.

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 Aß 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 pHi recovery from sustained intracellular acidosis (JH+ at pHi 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.