Modification of actin fibers changes the electrical phenotype of cardiac myofibroblasts

Background: Slow conduction and ectopic activity are major determinants of cardiac arrhythmogenesis. Both of these conditions can be elicited by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. MFBs appear during structural remodeling of the heart and are characterized by the expression of α-smooth muscle actin (α-SMA) containing stress fibers. In this study, we investigated whether pharmacological interference with the actin cytoskeleton affects myofibroblast arrhythmogeneicity. Methods: Experiments were performed with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse conduction velocity (θ) and maximal upstroke velocities of propagated action potentials (dV/dtmax), expressed as % action potential amplitude change (%APA) per ms, were measured optically using voltage sensitive dyes. Actin was destabilized by latrunculin B (LtB) and cytochalasin D and stabilized with jasplakinolide. Data are given as mean ± S.D. (n = 5-22). Single cell electrophysiology was assessed using standard patch-clamp techniques. Results: As revealed by immunocytochemistry, exposure of MFBs to LtB (0.01-10 μmol/L) profoundly disrupted stress fibers which led to drastic changes in cell morphology with MFBs assuming an astrocyte-like shape. In control cardiomyocyte strands (no MFB coat), LtB had negligible effects on θ and dV/dtmax. In contrast, LtB applied to MFB-coated strands increased θ dose-dependently from 197 ± 35 mm/s to 344 ± 26 mm/s and dV/dtmax from 38 ± 5 to 78 ± 3% APA/ms, i.e., to values virtually identical to those of cardiomyocyte control strands (339 ± 24 mm/s; 77 ± 3% APA/ms). Highly similar results were obtained when exposing the preparations to cytochalasin D. In contrast, stabilization of actin with increasing concentrations of jasplakinolide exerted no significant effects on impulse conduction characteristics in MFB-coated strands. Whole-cell patch-clamp experiments showed that LtB hyperpolarized MFBs from -25 mV to -50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause arrhythmogenic slow conduction and ectopic activity. Conclusion: Pharmacological interference with the actin cytoskeleton of cardiac MFBs affects their electrophysiological phenotype to such an extent that they loose their detrimental effects on cardiomyocyte electrophysiology. This result might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs

Resveratrol reduces myofibroblast arrhythmogenicity

Background: Among grape skin polyphenols, trans-resveratrol (RES) has been reported to slow the development of cardiac fibrosis and to affect myofibroblast (MFB) differentiation. Because MFBs induce slow conduction and ectopic activity following heterocellular gap junctional coupling to cardiomyocytes, we investigated whether RES and its main metabolites affect arrhythmogenic cardiomyocyte-MFB interactions. Methods: Experiments were performed with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage-sensitive dyes. Long-term video recordings served to characterize drug-related effects on ectopic activity. Data are given as means ± S.D. (n = 4–20). Results: Exposure of pure cardiomyocyte strands to RES at concentrations up to 10 µmol/L had no significant effects on impulse conduction velocity (θ) and maximal action potential upstroke velocities (dV/dtmax). By contrast, in MFB-coated strands exhibiting slow conduction, RES enhanced θ with an EC50 of ~10 nmol/L from 226 ± 38 to 344 ± 24 mm/s and dV/dtmax from 48 ± 7 to 69 ± 2%APA/ms, i.e., to values of pure cardiomyocyte strands (347 ± 33 mm/s; 75 ± 4%APA/ms). Moreover, RES led to a reduction of ectopic activity over the course of several hours in heterocellular preparations. RES is metabolized quickly in the body; therefore, we tested the main known metabolites for functional effects and found them similarly effective in normalizing conduction with EC50s of ~10 nmol/L (3-OH-RES), ~20 nmol/L (RES-3-O-β-glucuronide) and ~10 nmol/L (RES-sulfate), respectively. At these concentrations, neither RES nor its metabolites had any effects on MFB morphology and α-smooth muscle actin expression. This suggests that the antiarrhythmic effects observed were based on mechanisms different from a change in MFB phenotype. Conclusions: The results demonstrate that RES counteracts MFB-dependent arrhythmogenic slow conduction and ectopic activity at physiologically relevant concentrations. Because RES is rapidly metabolized following intestinal absorption, the finding of equal antiarrhythmic effectiveness of the main RES metabolites warrants their inclusion in future studies of potentially beneficial effects of these substances on the heart

The natural cardioprotective particle HDL modulates connexin43 gap junction channels

Aims High-density lipoprotein (HDL) is known for its cardioprotective properties independent from its cholesterol transport activity. These properties are mediated by activation of kinases such as protein kinase C (PKC). Connexin43 (Cx43) is a gap junction protein present in ventricular cardiomyocytes. PKC-dependent phosphorylation modifies Cx43 gap junction channel properties and is involved in cardioprotection. We hypothesized that cardioprotective properties of HDL may be mediated in part by affecting Cx43 gap junction channels. Methods and results Neonatal rat cardiomyocytes were treated with HDL and Cx43 phosphorylation was evaluated by western blotting and immunofluorescence. We found that HDL promoted phosphorylation of Cx43 with a maximal induction at 5 min, which was inhibited by pre-treatment with various PKC inhibitors. Sphingosine-1-phosphate (S1P), a component of HDL, induced effects that were similar to those of HDL. These compounds significantly reduced diffusion of fluorescent dye among cardiomyocytes (∼50%) which could be prevented by PKC inhibition. As observed during optical recordings of transmembrane voltage, HDL and S1P depressed impulse conduction only minimally (<5%). Moreover, 5 min of HDL and S1P treatment at the onset of reperfusion significantly reduced infarct size (∼50%) in response to 30 min ischaemia in ex vivo experiments. Conclusion Short-term treatment with HDL or S1P induces phosphorylation of Cx43 by a PKC-dependent pathway. HDL-induced phosphorylation of Cx43 reduced the diffusion of large tracer molecules between cells, whereas impulse conduction was maintained. Moreover, 5 min treatment with HDL confers cardioprotection against ischaemia/reperfusion injury. These results link Cx43 for the first time to the short-term cardioprotective effects of HD

Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of α-smooth muscle actin containing stress fibers

Rationale: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes. Objective: We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast–cardiomyocyte cross-talk. Methods and Results: Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. Conclusions: The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs

Estrategias y experiencias en la construcción de marca país en América del Sur

La construcción de marca país en América del Sur se encuentra en su etapa introductoria. Los países han creado marcas territoriales con restricciones presupuestarias con el finde proyectar una imagen integradora y positiva en los mercados internacionales. El desarrollo de una marca país y su gestión para aumentar los ingresos de exportación, atraer inversionistas e incentivar el turismo es una estrategia aún lejana para los países de América del Sur. Algunos países en América del Sur han hecho esfuerzos para mejorar su reputación, que se ha construido con posicionamientos no planeados, los intereses y opiniones de algunos líderes de opinión, la estabilidad e inestabilidad política y económica, y las transformaciones productivas. El siguiente artículo presenta los resultados de un estudio exploratorio y casuístico sobre las estrategias y experiencias de las marcas país en América del Sur. La hipótesis del estudio se enmarca en señalar que la construcción de marca país en América del Sur está en su fase introductoria y los resultados esperados no son consecuentes con la estrategia de crecimiento definida en los planes estratégicos de desarrollo de marca país. Del estudio se concluye que la formulación de una estrategia de marca país en América del Sur se ha vinculado a la promoción turística del país y no ha sido suficientemente asertiva en el interés de capitalizar su reputación en mercados internacionales.Strategies and Experiences in the Construction of Nation Brand in South America. The construction of country brands in South America is in the introductory stage. These countries have created territory brands with budgetary restrictions with the objective to project an integrative and positive image in the international markets. The development of a country brand to increase the export incomes, attract investors and encourage tourism is a very far strategy for South American countries. Many countries made qualified efforts to improve their reputation. Reputation originated by not planned positioning, interests and opinions of opinion leaders, political and economic stability or instability and productive transformations. This paper intends to show the results of an exploratory and casuistic study of the experiences and perceptions of brand countries in South America. The hypothesis of the study is that the brand country construction of South American countries is in the introductory stage and that there is no consistency between the results and their growing strategies for the development of their nation brands. From this study is possible to conclude that the design of a nation brand strategy in South America is linked to the touristic promotion of a country and it is not enough assertive to capitalize its reputation in the international markets.https://orcid.org/0000-0003-0001-9898https://www.scopus.com/authid/detail.uri?authorId=5685044010

Estrategias y experiencias en la construcción de marca país en América del Sur

La construcción de marca país en América del Sur se encuentra en su etapa introductoria. Los países han creado marcas territoriales con restricciones presupuestarias con el finde proyectar una imagen integradora y positiva en los mercados internacionales. El desarrollo de una marca país y su gestión para aumentar los ingresos de exportación, atraer inversionistas e incentivar el turismo es una estrategia aún lejana para los países de América del Sur. Algunos países en América del Sur han hecho esfuerzos para mejorar su reputación, que se ha construido con posicionamientos no planeados, los intereses y opiniones de algunos líderes de opinión, la estabilidad e inestabilidad política y económica, y las transformaciones productivas. El siguiente artículo presenta los resultados de un estudio exploratorio y casuístico sobre las estrategias y experiencias de las marcas país en América del Sur. La hipótesis del estudio se enmarca en señalar que la construcción de marca país en América del Sur está en su fase introductoria y los resultados esperados no son consecuentes con la estrategia de crecimiento definida en los planes estratégicos de desarrollo de marca país. Del estudio se concluye que la formulación de una estrategia de marca país en América del Sur se ha vinculado a la promoción turística del país y no ha sido suficientemente asertiva en el interés de capitalizar su reputación en mercados internacionales.Strategies and Experiences in the Construction of Nation Brand in South America. The construction of country brands in South America is in the introductory stage. These countries have created territory brands with budgetary restrictions with the objective to project an integrative and positive image in the international markets. The development of a country brand to increase the export incomes, attract investors and encourage tourism is a very far strategy for South American countries. Many countries made qualified efforts to improve their reputation. Reputation originated by not planned positioning, interests and opinions of opinion leaders, political and economic stability or instability and productive transformations. This paper intends to show the results of an exploratory and casuistic study of the experiences and perceptions of brand countries in South America. The hypothesis of the study is that the brand country construction of South American countries is in the introductory stage and that there is no consistency between the results and their growing strategies for the development of their nation brands. From this study is possible to conclude that the design of a nation brand strategy in South America is linked to the touristic promotion of a country and it is not enough assertive to capitalize its reputation in the international markets.https://orcid.org/0000-0003-0001-9898https://www.scopus.com/authid/detail.uri?authorId=5685044010