When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of ∼30% and in intracellular Na + concentration ([Na + ] i ) of ∼2.5 mmol l −1 over basal detected by H 2 DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na + ] i and the SFR, which were also blunted by Na + /H + exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na + ] i after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l −1 ) induced an increase in ·O 2 − production of ∼30-40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.

The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus). When embryonic-stem-cell-derived cardiomyocytes (ESC-CMs) are cultured on the substrates, biohybrids containing 32 wt% graphene significantly increase metabolic activity and cross-striated sarcomeric structures, indicative of the improved substrate suitability. By then applying electrical stimulation to these conductive biohybrid substrates, an enhancement of the alignment and maturation of the ESC-CMs is achieved. While this in vitro work has clearly shown the potential of these materials to be translated for cardiac applications, it is proposed that these graphene-based biohybrid platforms have potential for a myriad of other applications-particularly in electrically sensitive tissues, such as neural and neural and musculoskeletal tissues.

Abstract-Acute phosphodiesterase 5A inhibition by sildenafil or EMD360527/5 promoted profound inhibition of the cardiac Na ϩ /H ϩ exchanger (NHE-1), detected by the almost null intracellular pH recovery from an acute acid load (ammonium prepulse) in isolated papillary muscles from Wistar rats. Inhibition of phosphoglycerate kinase-1 (KT5823) restored normal NHE-1 activity, suggesting a causal link between phosphoglycerate kinase-1 increase and NHE-1 inhibition. We then tested whether the beneficial effects of NHE-1 inhibitors against the deleterious postmyocardial infarction (MI) remodeling can be detected after sildenafil-mediated NHE-1 inhibition. MI was induced by left anterior descending coronary artery ligation in Wistar rats, which were randomized to placebo or sildenafil (100 mg kg Ϫ1 day Ϫ1 ) for 6 weeks. Sildenafil significantly increased left ventricular phosphoglycerate kinase-1 activity in the post-MI group without affecting its expression. MI increased heart weight/body weight ratio, left ventricular myocyte cross-sectional area, interstitial fibrosis, and brain natriuretic peptide and NHE-1 expression. Sildenafil blunted these effects. Neither a significant change in infarct size nor a change in arterial or left ventricular systolic pressure was detected after sildenafil. MI decreased fractional shortening and the ratio of the maximum rate of rise of LVP divided by the pressure at the moment such maximum occurs, effects that were prevented by sildenafil. Intracellular pH recovery after an acid load was faster in papillary muscles from post-MI hearts (versus sham), whereas sildenafil significantly inhibited NHE-1 activity in both post-MI and sildenafil-treated sham groups. We conclude that increased phosphoglycerate kinase-1 activity after acute phosphodiesterase 5A inhibition blunts NHE-1 activity and protects the heart against post-MI remodeling and dysfunction.

Key points• Acid extrusion from ventricular myocytes typically occurs via Na + /H + exchange (NHE1) and Na + -HCO 3 − co-transporters (NBC). This maintains intracellular pH at ∼7.2: The membrane distribution of these transporters is uncertain.• Immunofluorescence indicates that: NBC isoforms are located in lateral sarcolemma, intercalated discs and transverse tubules, whereas NHE1 is densely expressed at intercalated discs.• Functional experiments with detubulated myocytes indicate reduced acid extrusion on NBC but no effect on NHE1 activity, confirming exclusion of NHE1 function from transverse tubules.• Stimulating NHE1 activity induces sub-sarcolemmal [H + ] i depletion (forming local pH i microdomains), particularly at intercalated discs, while stimulating NBC activity induces no pH i microdomains.• Our results provide the first demonstration that pH i in ventricular myocytes is locally controlled through selective trafficking of membrane ion transporters. NHE1 preferentially controls pH i at intercalated discs, where cell-to-cell gap-junctional channels are located, while NBC influences pH i adjacent to transverse tubules, where key proteins for excitation-contraction coupling are located.Abstract Membrane acid extrusion by Na + /H + exchange (NHE1) and Na + -HCO 3 − co-transport (NBC) is essential for maintaining a low cytoplasmic [H + ] (∼60 nM, equivalent to an intracellular pH (pH i ) of 7.2). This protects myocardial function from the high chemical reactivity of H + ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pH i regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 M formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pH i imaging reveals that NBC stimulation induces spatially uniform pH i recovery from acidosis, whereas NHE1 stimulation induces pH i non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pH i microdomains is favoured by low cytoplasmic H + mobility and long H + diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H + ] i regulation in ventricular myocytes,

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The aim of this work was to assess the possible correlation between oxidative damage and the development of cardiac hypertrophy in heart tissue from young (40-d-old) and older (4-, 11-and 19-month-old) spontaneously hypertensive rats (SHR) in comparison with age-matched Wistar (W) rats. To this end, levels of thiobarbituric acid reactive substances (TBARS), nitrotyrosine contents, NAD(P)H oxidase activity, superoxide production, and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were determined. Compared to age-matched normotensive rats, SHR showed a significant increase in systolic blood pressure from 40 d of age and left ventricular hypertrophy (LVH) was significantly evident from 4 months of age. W rats (11-and 19-month-old) also showed an increase in LVH with aging. TBARS and nitrotyrosine levels were similar in young rats from both strains and were significantly increased with age in both strains, with the values in SHR being significantly higher than those in age-matched W rats. NAD(P)H activity was similar in young SHR and W rats, whereas it was higher in aged SHR compared with age-matched W rats. Compared to W rats, superoxide production was higher in aged SHR, and was abolished by NAD(P)H inhibition with apocynin. CAT activity was increased in the hearts of 4-month-old SHR compared to age-matched W rats and was decreased in the hearts of the oldest SHR compared to the oldest W rats. SOD and GPx activities decreased in both rat strains with aging. Moreover, an increase in collagen deposition with aging was evident in both rat strains. Taken together, these data showed that aged SHR exhibited higher cardiac hypertrophy and oxidative damage compared to W rats, indicating that the two undesirable effects are associated. That is, oxidative stress appears to be a cause and/or consequence of hypertrophy development in this animal model. (Hypertens Res 2008; 31: 1465-1476)

Abstract-The effect of endurance training (swimming 90 min/d for 5 days a week for 60 days) on cardiac hypertrophy was investigated in the spontaneously hypertensive rat (SHR). Sedentary SHRs (SHR-Cs) and normotensive Wistar rats were used as controls. Exercise training enhanced myocardial hypertrophy assessed by left ventricular weight/tibial length (228Ϯ7 versus 251Ϯ5 mg/cm in SHR-Cs and exercised SHRs [SHR-Es], respectively). Myocyte cross-sectional area increased Ϸ40%, collagen volume fraction decreased Ϸ50%, and capillary density increased Ϸ45% in SHR-Es compared with SHR-Cs. The mRNA abundance of atrial natriuretic factor and myosin light chain 2 was decreased by the swimming routine (100Ϯ19% versus 41Ϯ10% and 100Ϯ8% versus 61Ϯ9% for atrial natriuretic factor and myosin light chain 2 in SHR-Cs and SHR-Es, respectively). The expression of sarcoplasmic reticulum Ca 2ϩ pump was significantly augmented, whereas that of Na ϩ /Ca 2ϩ exchanger was unchanged (93Ϯ7% versus 167Ϯ8% and 158Ϯ13% versus 157Ϯ7%, sarcoplasmic reticulum Ca 2ϩ pump and Na ϩ /Ca 2ϩ exchanger in SHR-Cs and SHR-Es, respectively; PϽ0.05). Endurance training inhibited apoptosis, as reflected by a decrease in caspase 3 activation and poly(ADP-ribose) polymerase-1 cleavage, and normalized calcineurin activity without inducing significant changes in the phosphatidylinositol 3-kinase/Akt pathway. The swimming routine improved midventricular shortening determined by echocardiography (32.4Ϯ0.9% versus 36.9Ϯ1.1% in SHR-Cs and SHR-Es, respectively; PϽ0.05) and decreased the left ventricular free wall thickness/left ventricular cavity radius toward an eccentric model of cardiac hypertrophy (0.59Ϯ0.02 versus 0.53Ϯ0.01 in SHR-Cs and SHR-Es, respectively; PϽ0.05). In conclusion, we present data demonstrating the effectiveness of endurance training to convert pathological into physiological hypertrophy improving cardiac performance. The reduction of myocardial fibrosis and calcineurin activity plus the increase in capillary density represent factors to be considered in determining this beneficial effect.