Intramyocardial gene silencing by interfering RNA

RNAi is a widely used methodology for gene silencing. The action mechanism of siRNA molecules has been well studiedin recent years, and the technique has been optimized in terms of safety and effectiveness. Cardiovascular diseases havea high incidence in the current population, and despite of the extensive research, safe and efficient therapeutics have notyet been found, which is reflected by 17.1 million people who die each year for this cause. In this context, siRNAs arebeing considered a therapeutic tool to regulate the expression of genes involved in the generation of these pathologies.The efficacy of siRNAs entry to cardiomyocytes, the safety of the delivery process and the degree of silencing achievedare main aspects before consider it as a cardiovascular disease therapy. Presently, we will give a brief outline of thecurrent understanding of the RNAi mechanism and the delivery system to the heart. We describe the use of lentivirus fora functional silencing of cardiac proteins in the study of a pathophysiological process, the slow force response to cardiacstretch.Fil: Brea, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Morgan, Patricio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Perez, Nestor Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; Argentin

Regulación de los canales de calcio por dihidropiridinas en corazón de rata

De acuerdo con los resultados de la bibliografía, los efectos obtenidos sobre la función cardíaca y sobre las características del canal L de Ca2+ luego del tratamiento crónico con nifedipina son variados y contradictorios (43,56,99,100). Teniendo en cuenta que los canales L de Ca2+ constituyen la primera etapa en la génesis de la contracción, en este estudio se evaluó el efecto del tratamiento crónico con nifedipina sobre las características y funcionalidad de los canales L de Ca2+ en el corazón de rata. Con este propósito se analizó: • La densidad de los sitios receptores de dihidropiridinas en membranas cardíacas. • El “transient” de Ca2+ y el acortamiento celular en miocitos aislados.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Regulación de los canales de calcio por dihidropiridinas en corazón de rata

De acuerdo con los resultados de la bibliografía, los efectos obtenidos sobre la función cardíaca y sobre las características del canal L de Ca2+ luego del tratamiento crónico con nifedipina son variados y contradictorios (43,56,99,100). Teniendo en cuenta que los canales L de Ca2+ constituyen la primera etapa en la génesis de la contracción, en este estudio se evaluó el efecto del tratamiento crónico con nifedipina sobre las características y funcionalidad de los canales L de Ca2+ en el corazón de rata. Con este propósito se analizó: • La densidad de los sitios receptores de dihidropiridinas en membranas cardíacas. • El “transient” de Ca2+ y el acortamiento celular en miocitos aislados.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na⁺ independent. Studies in oocytes and HEK293 cells could not detect Na⁺⁻ coupled HCO3⁻ transport or Cl⁻/HCO3⁻ exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltagedependent OH⁻ or H⁺ movement was not measurable in SLC4A11- expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH⁻/H⁺ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H⁺.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Intramyocardial gene silencing by interfering RNA

RNAi is a widely used methodology for gene silencing. The action mechanism of siRNA molecules has been well studiedin recent years, and the technique has been optimized in terms of safety and effectiveness. Cardiovascular diseases have a high incidence in the current population, and despite of the extensive research, safe and efficient therapeutics have notyet been found, which is reflected by 17.1 million people who die each year for this cause. In this context, siRNAs arebeing considered a therapeutic tool to regulate the expression of genes involved in the generation of these pathologies.The efficacy of siRNAs entry to cardiomyocytes, the safety of the delivery process and the degree of silencing achievedare main aspects before consider it as a cardiovascular disease therapy. Presently, we will give a brief outline of thecurrent understanding of the RNAi mechanism and the delivery system to the heart. We describe the use of lentivirus fora functional silencing of cardiac proteins in the study of a pathophysiological process, the slow force response to cardiac stretch.Centro de Investigaciones Cardiovasculare

Epidermal growth factor receptor silencing blunts the slow force response to myocardial stretch

Background-Myocardial stretch increases force biphasically: the Frank-Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]-EGFR). Methods and Results-Silencing capability of p-shEGFR was assessed in EGFR-GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA-EGFR-injected hearts showed -60% reduction of EGFR protein expression compared with shRNA-SCR-injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA-SCR group, while it was blunted in shRNA-EGFR-expressing muscles. Angiotensin II (Ang II)-dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate-free medium, demonstrating an increase in shRNA-SCR-injected myocardium, an effect not observed in the silenced group. Ang II- or EGF-triggered reactive oxygen species production was significantly reduced in shRNA-EGFR-injected hearts compared with that in the shRNA-SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox-sensitive pathway, leading to p90RSK activation in shRNA-SCR-injected myocardium, an effect that was absent in the shRNA-EGFR group. Conclusions-Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox-sensitive kinase activation pathway, leading to SFR development.Centro de Investigaciones CardiovascularesFacultad de Ciencias Veterinaria

Hyperactivity and altered mRNA isoform expression of the Cl¯/HCO3¯ anion-exchanger in the hypertrophied myocardium

Objective: The aim was to examine the regulation of the cardiac Na+-independent Cl¯/HCO3¯ exchanger (AE) mRNA isoform expression in association to the enhanced AE activity in the hypertrophied myocardium of spontaneously hypertensive rats (SHR). Methods: AE activity was determined by the initial rates of the pHi recovery from imposed intracellular alkalinization (forward mode of exchange) and the pHi rise induced by Cl¯ removal (reverse mode). Net HCO3¯ (JHCO3¯) efflux and influx were respectively determined. AE mRNA isoforms were analyzed by Northern blot with specific probes to detect AE1, AE2 and AE3 mRNAs. Results: Initial JHCO3¯ efflux after imposed alkaline load (pHi≅7.5) was higher in SHR than in normotensive WKY rats (3.01±0.33, n=7, vs. 0.64±0.29 mM/min, n=5, PHCO3¯ influx induced by Cl¯ deprivation was also increased in SHR, 4.24±0.56 mM/min (n=10) versus 2.31±0.26 (n=10, P<0.05) in WKY. In arbitrary units, the 4.1-kb AE1 mRNA decreased in SHR (0.15±0.01, n=7) compared to WKY (0.29±0.06, n=7, P<0.05), whereas the 3.6-kb mRNA did not change. AE2 mRNAs were similarly expressed in WKY and SHR. Cardiac specific AE3 (cAE3) mRNA decreased in SHR, 1.10±0.16 arbitrary units (n=8) versus 1.79±0.24, (n=8, P<0.05) in WKY. Full length AE3 (flAE3) mRNA increased from 0.69±0.06 (WKY, n=8) to 1.25±0.19 arbitrary units in SHR (n=8, P<0.05). Conclusions: The increase in flAE3 mRNA expression in cardiac tissue from the SHR is an adaptive change of the hypertrophied myocardium that might be in connection with the increased activity of the AE.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium

Mineralocorticoid receptor (MR) antagonists decreasemorbidity andmortality in heart failure patients forwhom oxidative stress is usual; however, the underlying mechanism for this protection is unclear. Since aldosterone stimulates reactive oxygen species (ROS) production in several tissues,we explored its effect and the intracellular pathway involved in the rat myocardium. Aldosterone dose-dependently increased O2 − production inmyocardial slices. At 10 nmol/L, aldosterone increased O2 − to 165 ± 8.8% of control, an effect prevented not only by the MR antagonists eplerenone and spironolactone (107 ± 7.8 and 103 ± 5.3%, respectively) but also by AG1478 (105 ± 8.0%), antagonist of the EGF receptor (EGFR). Similar results were obtained by silencing MR expression through the direct intramyocardial injection of a lentivirus coding for a siRNA against the MR. The aldosterone effect on O2 − production was mimicked by the mKATP channel opener diazoxide and blocked by preventing its opening with 5-HD and glibenclamide, implicating the mitochondria as the source of O2 −. Inhibiting the respiratory chainwith rotenone or mitochondrial permeability transition (MPT)with cyclosporine A or bongkrekic acid also canceled aldosterone-induced O2 − production. In addition, aldosterone effect depended on NADPH oxidase and phosphoinositide 3-kinase activation, as apocynin and wortmannin, respectively, inhibited it. EGF (0.1 μg/ mL) similarly increased O2 −, although in this case MR antagonists had no effect, suggesting that EGFR transactivation occurred downstream from MR activation. Inhibition of mKATP channels, the respiratory chain, or MPT did not prevent Akt phosphorylation, supporting that it happened upstreamof the mitochondria. Importantly, cardiomyocytes were confirmed as a source of aldosterone induced mitochondrial ROS production in experiments performed in isolated cardiac myocytes. These results allowus to speculate that the beneficial effects ofMRantagonists in heart failure may be related to a decrease in oxidative stress.Centro de Investigaciones Cardiovasculare

Epidermal growth factor receptor silencing blunts the slow force response to myocardial stretch

Background-Myocardial stretch increases force biphasically: the Frank-Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]-EGFR). Methods and Results-Silencing capability of p-shEGFR was assessed in EGFR-GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA-EGFR-injected hearts showed -60% reduction of EGFR protein expression compared with shRNA-SCR-injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA-SCR group, while it was blunted in shRNA-EGFR-expressing muscles. Angiotensin II (Ang II)-dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate-free medium, demonstrating an increase in shRNA-SCR-injected myocardium, an effect not observed in the silenced group. Ang II- or EGF-triggered reactive oxygen species production was significantly reduced in shRNA-EGFR-injected hearts compared with that in the shRNA-SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox-sensitive pathway, leading to p90RSK activation in shRNA-SCR-injected myocardium, an effect that was absent in the shRNA-EGFR group. Conclusions-Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox-sensitive kinase activation pathway, leading to SFR development.Centro de Investigaciones CardiovascularesFacultad de Ciencias Veterinaria

Hyperactivity and altered mRNA isoform expression of the Cl¯/HCO3¯ anion-exchanger in the hypertrophied myocardium

Objective: The aim was to examine the regulation of the cardiac Na+-independent Cl¯/HCO3¯ exchanger (AE) mRNA isoform expression in association to the enhanced AE activity in the hypertrophied myocardium of spontaneously hypertensive rats (SHR). Methods: AE activity was determined by the initial rates of the pHi recovery from imposed intracellular alkalinization (forward mode of exchange) and the pHi rise induced by Cl¯ removal (reverse mode). Net HCO3¯ (JHCO3¯) efflux and influx were respectively determined. AE mRNA isoforms were analyzed by Northern blot with specific probes to detect AE1, AE2 and AE3 mRNAs. Results: Initial JHCO3¯ efflux after imposed alkaline load (pHi≅7.5) was higher in SHR than in normotensive WKY rats (3.01±0.33, n=7, vs. 0.64±0.29 mM/min, n=5, PHCO3¯ influx induced by Cl¯ deprivation was also increased in SHR, 4.24±0.56 mM/min (n=10) versus 2.31±0.26 (n=10, P<0.05) in WKY. In arbitrary units, the 4.1-kb AE1 mRNA decreased in SHR (0.15±0.01, n=7) compared to WKY (0.29±0.06, n=7, P<0.05), whereas the 3.6-kb mRNA did not change. AE2 mRNAs were similarly expressed in WKY and SHR. Cardiac specific AE3 (cAE3) mRNA decreased in SHR, 1.10±0.16 arbitrary units (n=8) versus 1.79±0.24, (n=8, P<0.05) in WKY. Full length AE3 (flAE3) mRNA increased from 0.69±0.06 (WKY, n=8) to 1.25±0.19 arbitrary units in SHR (n=8, P<0.05). Conclusions: The increase in flAE3 mRNA expression in cardiac tissue from the SHR is an adaptive change of the hypertrophied myocardium that might be in connection with the increased activity of the AE.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare