Carolina D. Garciarena

Endurance Training in the Spontaneously Hypertensive Rat: Conversion of Pathological into Physiological Cardiac Hypertrophy

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 (228±7 versus 251±5 mg/cm in SHR-Cs and exercised SHRs [SHR-Es], respectively). Myocyte cross-sectional area increased ≈40%, collagen volume fraction decreased ≈50%, and capillary density increased ≈45% 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 (100±19% versus 41±10% and 100±8% versus 61±9% for atrial natriuretic factor and myosin light chain 2 in SHR-Cs and SHR-Es, respectively). The expression of sarcoplasmic reticulum Ca2+ pump was significantly augmented, whereas that of Na+/Ca2+ exchanger was unchanged (93±7% versus 167±8% and 158±13% versus 157±7%, sarcoplasmic reticulum Ca2+ pump and Na+/Ca2+ exchanger in SHR-Cs and SHR-Es, respectively; P<0.05). 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 (32.4±0.9% versus 36.9±1.1% in SHR-Cs and SHR-Es, respectively; P<0.05) and decreased the left ventricular free wall thickness/left ventricular cavity radius toward an eccentric model of cardiac hypertrophy (0.59±0.02 versus 0.53±0.01 in SHR-Cs and SHR-Es, respectively; P<0.05). 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.

The Positive Inotropic Effect of Angiotensin II: Role of Endothelin-1 and Reactive Oxygen Species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (&Dgr;ROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II–induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II–treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.

Mitochondrial reactive oxygen species activate the slow force response to stretch in feline myocardium

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 H2DCFDA 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 ·O2− 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.

Phosphodiesterase 5A Inhibition Induces Na/H Exchanger Blockade and Protection Against Myocardial Infarction

Acute phosphodiesterase 5A inhibition by sildenafil or EMD360527/5 promoted profound inhibition of the cardiac Na+/H+ 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−1 day−1) 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.

Is Cardiac Hypertrophy in Spontaneously Hypertensive Rats the Cause or the Consequence of Oxidative Stress

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.

Sarcolemmal localisation of Na+/H+ exchange and Na+–HCO3− co‐transport influences the spatial regulation of intracellular pH in rat ventricular myocytes

•  Acid extrusion from ventricular myocytes typically occurs via Na+/H+ exchange (NHE1) and Na+–HCO3− 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 pHi microdomains), particularly at intercalated discs, while stimulating NBC activity induces no pHi microdomains. •  Our results provide the first demonstration that pHi in ventricular myocytes is locally controlled through selective trafficking of membrane ion transporters. NHE1 preferentially controls pHi at intercalated discs, where cell‐to‐cell gap‐junctional channels are located, while NBC influences pHi adjacent to transverse tubules, where key proteins for excitation–contraction coupling are located.

Early signals after stretch leading to cardiac hypertrophy. Key role of NHE-1.

The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCX(rev)), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.

Myocardial Reperfusion Injury: Reactive Oxygen Species vs. NHE-1 Reactivation

Background/Aims: Flow restoration to ischemic myocardium reduces infarct size (IS), but it also promotes reperfusion injury. A burst of reactive oxygen species (ROS) and/or NHE-1 reactivation were proposed to explain this injury. Our study was aimed to shed light on this unresolved issue. Methods: Regional infarction (40 min-ischemia/2 hs-reperfusion) was induced in isolated and perfused rat hearts. Maximal doses of N-(2-mercaptopropionyl)-glycine (MPG 2mmol/L, ROS scavenger), cariporide (10µmol/L, NHE-1 inhibitor), or sildenafil (1µmol/L, phosphodiesterase5A inhibitor) were applied at reperfusion onset. Their effects on IS, myocardial concentration of thiobarbituric acid reactive substances (TBARS), ERK1/2, p90RSK, and NHE-1 phosphorylation were analyzed. Results: All treatments decreased IS ∼ 50% vs. control. No further protection was obtained by combining cariporide or MPG with sildenafil. Myocardial TBARS increased after infarction and were decreased by MPG or cariporide, but unaffected by sildenafil. In line with the fact that ROS induce MAPK-mediated NHE-1 activation, myocardial infarction increased ERK1/2, p90RSK, and NHE-1 phosphorylation. MPG and cariporide cancelled these effects. Sildenafil did not reduce the phosphorylated ERK1/2-p90RSK levels but blunted NHE-1 phosphorylation suggesting a direct dephosphorylating action. Conclusions: 1) Reperfusion injury would result from ROS-triggered MAPK-mediated NHE-1 phosphorylation (and reactivation) during reperfusion; 2) sildenafil protects the myocardium by favouring NHE-1 dephosphorylation and bypassing ROS generation.

Decreased Activity of the Na+/H+ Exchanger by Phosphodiesterase 5A Inhibition Is Attributed to an Increase in Protein Phosphatase Activity

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na+/H+ exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 &mgr;mol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (JH; in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 &mgr;mol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal–regulated kinase 1/2-p90RSK pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1–dependent reduction in NHE-1 phosphorylation.

Towards 3D in vitro models for the study of cardiovascular tissues and disease

The field of tissue engineering is developing biomimetic biomaterial scaffolds that are showing increasing therapeutic potential for the repair of cardiovascular tissues. However, a major opportunity exists to use them as 3D in vitro models for the study of cardiovascular tissues and disease in addition to drug development and testing. These in vitro models can span the gap between 2D culture and in vivo testing, thus reducing the cost, time, and ethical burden of current approaches. Here, we outline the progress to date and the requirements for the development of ideal in vitro 3D models for blood vessels, heart valves, and myocardial tissue.