Papers by María Pura Hortigón Vinagre
International Journal of Molecular Sciences, 2022
Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results i... more Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results in reducing hyperglycaemia and insulin resistance. However, diabetic patients have higher risk of ventricular arrhythmia and sudden cardiac death, and metformin failed to reduce ventricular arrhythmia in clinical trials. In order to explore the mechanisms responsible for the lack of protective effect, we investigated in vivo the effect of metformin on cardiac electrical activity in non-diabetic rats; and in vitro in isolated ventricular myocytes, HEK293 cells expressing the hERG channel and human induced pluripotent stem cells derived cardiomyocytes (hIPS-CMs). Surface electrocardiograms showed that long-term metformin treatment (7 weeks) at therapeutic doses prolonged cardiac repolarization, reflected as QT and QTc interval duration, and increased ventricular arrhythmia during the caffeine/dobutamine challenge. Patch-clamp recordings in ventricular myocytes isolated from treated animals showed that the cellular mechanism is a reduction in the cardiac transient outward potassium current (I to). In vitro, incubation with metformin for 24 h also reduced I to , prolonged action potential duration, and increased spontaneous contractions in ventricular myocytes isolated from control rats. Metformin incubation also reduced I hERG in HEK293 cells. Finally, metformin incubation prolonged action potential duration at 30% and 90% of repolarization in hIPS-CMs, which is compatible with the reduction of I to and I hERG. Our results show that metformin directly modifies the electrical behavior of the normal heart. The mechanism consists in the inhibition of repolarizing currents and the subsequent decrease in repolarization capacity, which prolongs AP and QTc duration.
Bioengineering, 2022
In this work, we show that valve-based bioprinting induces no measurable detrimental effects on h... more In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was threefold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalvebased cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell 2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell 2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.
International Journal of Molecular Sciences, 2021
The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiC... more The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (I K1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-I K1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-I K1 :hiCM ratios was compared with co-cultures using wildtype (HEK-WT:hiCM) or hiCM alone on days 3-8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that I K1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK-WT. The addition of the I K1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-I K1 :hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of I K1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient I K1 was integrated into the syncytium.
Journal of Pharmacological and Toxicological Methods, 2017
Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging ... more Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as new and human-relevant source in vitro model for cardiac safety assessment that allow us to investigate a set of 20 reference drugs for predicting cardiac arrhythmogenic liability using optical action potential (oAP) assay. Methods: Here, we describe our examination of the oAP measurement using a voltage sensitive dye (Di-4-ANEPPS) to predict adverse compound effects using hiPS-CMs and 20 cardioactive reference compounds. Fluorescence signals were digitized at 10 kHz and the records subsequently analyzed off-line. Cells were exposed to 30 min incubation to vehicle or compound (n = 5/dose, 4 doses/compound) that were blinded to the investigating laboratory. Action potential parameters were measured, including rise time (T rise) of the optical action potential duration (oAPD). Results: Significant effects on oAPD were sensitively detected with 11 QT-prolonging drugs, while oAPD shortening was observed with I Ca-antagonists, I Kr-activator or ATP-sensitive K + channel (K ATP)-opener. Additionally, the assay detected varied effects induced by 6 different sodium channel blockers. The detection threshold for these drug effects was at or below the published values of free effective therapeutic plasma levels or effective concentrations by other studies. Discussion: The results of this blinded study indicate that OAP is a sensitive method to accurately detect druginduced effects (i.e., duration/QT-prolongation, shortening, beat rate, and incidence of early after depolarizations) in hiPS-CMs; therefore, this technique will potentially be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.
Frontiers in Physiology, 2017
Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in diseas... more Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.
British Journal of Pharmacology, 2017
Human ether-a-go-go-related gene (hERG; Kv11.1) channel inhibition is a widely accepted predictor... more Human ether-a-go-go-related gene (hERG; Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG
channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives
to investigate the relationship between standard measures of hERG current block in an expression system and changes in
action potential duration (APD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The interference
from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model.
Toxicological Sciences, 2017
Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-rela... more Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation-contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways.
Toxicological Sciences, 2017
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of dru... more Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-ago go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC
ACS Biomaterials Science and Engineering, 2016
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities fo... more Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities for cardiac research but they exhibit an immature phenotype, which influences experimental outcomes. The aim of our research is to investigate the effects of a topographical gradient substrate on the morphology and function of commercially available hiPSC-CM. The lateral dimensions the microgrooves on the substrate varied from 8 to 100 μm space between the 8 μm grooves on one axis and from ∼5 nm to ∼1 μm in depth on the other axis. Cells were seeded homogeneously across the substrate and according to the manufacturers protocols. At days 4 and 10, measures of eccentricity, elongation, orientation, sarcomere length (SL), and contractility of the hiPSC-CM were taken. Only the deepest and widest region (8−30 μm wide and 0.85−1 μm deep) showed a significantly higher percentage of hiPSC-CM with an increased eccentricity (31.3 ± 6.4%), elongation (10.4 ± 4.3%), and orientation (<10°) (32.1 ± 2.7%) when compared with the control (flat substrate) (15.8 ± 5.0%, 3.4 ± 2.7%, and 10.6 ± 1.1%, respectively). Additionally, during stimulus-induced contraction, the relaxation phase of the twitch was prolonged (400 ms) compared to nonelongated cells (200 ms). These findings support the potential use of dual microgradient substrates to investigate substrate topographies that stimulate migration and/or maturation of hiPSC-CM.
Journal of the American College of Cardiology, 2016
BACKGROUND Angiotensin-(1-9) [Ang-(1-9)] is a novel peptide of the counter-regulatory axis of the... more BACKGROUND Angiotensin-(1-9) [Ang-(1-9)] is a novel peptide of the counter-regulatory axis of the reninangiotensin-aldosterone system previously demonstrated to have therapeutic potential in hypertensive cardiomyopathy when administered via osmotic mini-pump. Here, we investigate whether gene transfer of Ang-(1-9) is cardioprotective in a murine model of myocardial infarction (MI). OBJECTIVES The authors evaluated effects of Ang-(1-9) gene therapy on myocardial structural and functional remodeling post-infarction. METHODS C57BL/6 mice underwent permanent left anterior descending coronary artery ligation and cardiac function was assessed using echocardiography for 8 weeks followed by a terminal measurement of left ventricular pressure volume loops. Ang-(1-9) was delivered by adeno-associated viral vector via single tail vein injection immediately following induction of MI. Direct effects of Ang-(1-9) on cardiomyocyte excitation/contraction coupling and cardiac contraction were evaluated in isolated mouse and human cardiomyocytes and in an ex vivo Langendorff-perfused whole-heart model. RESULTS Gene delivery of Ang-(1-9) reduced sudden cardiac death post-MI. Pressure volume measurements revealed complete restoration of end-systolic pressure, ejection fraction, end-systolic volume, and the end-diastolic pressure volume relationship by Ang-(1-9) treatment. Stroke volume and cardiac output were significantly increased versus sham. Histological analysis revealed only mild effects on cardiac hypertrophy and fibrosis, but a significant increase in scar thickness. Direct assessment of Ang-(1-9) on isolated cardiomyocytes demonstrated a positive inotropic effect via increasing calcium transient amplitude and contractility. Ang-(1-9) increased contraction in the Langendorff model through a protein kinase A-dependent mechanism. CONCLUSIONS Our novel findings showed that Ang-(1-9) gene therapy preserved left ventricular systolic function post-MI, restoring cardiac function. Furthermore, Ang-(1-9) directly affected cardiomyocyte calcium handling through a protein kinase A-dependent mechanism. These data emphasized Ang-(1-9) gene therapy as a potential new strategy in the context of MI.
Toxicological Sciences, 2016
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platf... more Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD 90 : 2296 15 ms vs 427 6 49 ms [mean 6 SD, P < 0.01]; average spontaneous cycle length: 0.99 6 0.02 s vs 1.47 6 0.35 s [mean 6 SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10-90% rise time of the AP (T rise) was $6 ms and was normally distributed when expressed as 1/T 2 rise in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.
FEBS Open Bio, 2014
Phosphorylated heat shock protein 20 (HSP20) is cardioprotective. Using human induced pluripotent... more Phosphorylated heat shock protein 20 (HSP20) is cardioprotective. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model of pressure overload mediated hypertrophy, we show that peptide disruption of the HSP20-phosphodiesterase 4D (PDE4D) complex results in attenuation of action potential prolongation and protection against adverse cardiac remodelling. The later was evidenced by improved contractility, decreased heart weight to body weight ratio, and reduced interstitial and perivascular fibrosis. This study demonstrates that disruption of the specific HSP20-PDE4D interaction leads to attenuation of pathological cardiac remodelling.
Planta Medica, 2014
An interdisciplinary experimental investigation
on the antioxidant activity of Thymbra capitata e... more An interdisciplinary experimental investigation
on the antioxidant activity of Thymbra capitata essential
oilwas made. This plant is a Mediterranean
culinary herb, whose essential oil antioxidant
power has recently been demonstrated in vitro as
one of the highest in nature. We tested if this in
vitro antioxidant capacity was reproducible on
biological systems using as model system primary
cultures of neonatal rat cardiomyocytes treated
with the lipid peroxidation product 4-hydroxy-2-
nonenal. The composition and the in vitro antioxidant
activity of the T. capitata essential oil were
also assessed. Cell viability, mitochondrial membrane
potential, and reactive oxygen species level
were measured in cells treated with pathophysiologic
doses of 4-hydroxy-2-nonenal (< 10 μM) or
vehicle after being pre-incubated with small concentrations
of the T. capitata essential oil, and the
ability of small doses (< 40 ppm) to prevent the
death of neonatal rat cardiomyocytes proved very
remarkable. Long-term pre-incubation (12 h)
with 20 ppm prevented 4-hydroxy-2-nonenal-induced
cell death and avoided mitochondrial
membrane potential loss and reactive oxygen species
generation caused by 4-hydroxy-2-nonenal.
A deleterious effect was shown at doses higher
than 40 ppm. The results of this study pave the
way to further analysis in animal models to
achieve a deeper understanding of the in vivo
antioxidant power of T. capitata essential oil.
Cardiovascular Toxicology, 2014
Lipid peroxidation (LP), induced by oxidative stress, is associated with degenerative processes. ... more Lipid peroxidation (LP), induced by oxidative stress, is associated with degenerative processes. 4-Hydroxy-2-nonenal (HNE), a highly reactive diffusible product of LP, is considered by-product and mediator of oxidative stress. Its level increases under pathological conditions such as cardiovascular diseases. In this study, we partially characterized the mechanisms of HNE-mediated cytotoxicity in cardiomyocytes. After establishing that pathophysiological doses of HNE trigger cell death dependent on the incubation time and dose of HNE (LD 50 = 4.4 lM), we tackled the mechanisms that underlie the cell death induced by HNE. Our results indicate that HNE rapidly increases intracellular Ca 2? ; it also increases the rate of reactive oxygen species generation and causes a loss of mitochondrial membrane potential (DWm) as well as a decrease in the ATP and GSH levels. Such alterations result in the activation of caspase-3 and DNA breakdown, both characteristic features of apoptotic cell death, as well as disruption of the cytoskeleton. Moreover, the nucleophilic compounds N-acetyl-cysteine and b-mercapto-propionyl-glycine, and the synthetic antioxidant Trolox exert a potent antioxidant action against HNE damage; this suggests its use as effective compounds in order to reduce the damage occurred as consequence of cardiovascular disorders in which oxidative stress and hence LP take place.
Free Radical Biology and Medicine, 2011
Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition ... more Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the
maximal ATPase activity and Ca2+ transport ability of SERCA1a, the Ca2+ pump in these membranes. The
concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding
and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca2+ binding to
the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were
identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also
reacted with Lys515 within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the
steady-state ability of SR vesicles to sequester Ca2+ and the ATPase activity of SR membranes in the absence of
added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected
the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNEtreated
SR vesicles to Ca2+, an increase in permeability that did not arise from alteration of the lipid
component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent
increase in permeability probably arose from modification of proteins of about 150–160 kDa, present in very
low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae).
HNE-induced promotion, via these proteins, of Ca2+ leakage pathways might be involved in the general toxic
effects of HNE.
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Papers by María Pura Hortigón Vinagre
channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives
to investigate the relationship between standard measures of hERG current block in an expression system and changes in
action potential duration (APD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The interference
from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model.
on the antioxidant activity of Thymbra capitata essential
oilwas made. This plant is a Mediterranean
culinary herb, whose essential oil antioxidant
power has recently been demonstrated in vitro as
one of the highest in nature. We tested if this in
vitro antioxidant capacity was reproducible on
biological systems using as model system primary
cultures of neonatal rat cardiomyocytes treated
with the lipid peroxidation product 4-hydroxy-2-
nonenal. The composition and the in vitro antioxidant
activity of the T. capitata essential oil were
also assessed. Cell viability, mitochondrial membrane
potential, and reactive oxygen species level
were measured in cells treated with pathophysiologic
doses of 4-hydroxy-2-nonenal (< 10 μM) or
vehicle after being pre-incubated with small concentrations
of the T. capitata essential oil, and the
ability of small doses (< 40 ppm) to prevent the
death of neonatal rat cardiomyocytes proved very
remarkable. Long-term pre-incubation (12 h)
with 20 ppm prevented 4-hydroxy-2-nonenal-induced
cell death and avoided mitochondrial
membrane potential loss and reactive oxygen species
generation caused by 4-hydroxy-2-nonenal.
A deleterious effect was shown at doses higher
than 40 ppm. The results of this study pave the
way to further analysis in animal models to
achieve a deeper understanding of the in vivo
antioxidant power of T. capitata essential oil.
maximal ATPase activity and Ca2+ transport ability of SERCA1a, the Ca2+ pump in these membranes. The
concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding
and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca2+ binding to
the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were
identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also
reacted with Lys515 within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the
steady-state ability of SR vesicles to sequester Ca2+ and the ATPase activity of SR membranes in the absence of
added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected
the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNEtreated
SR vesicles to Ca2+, an increase in permeability that did not arise from alteration of the lipid
component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent
increase in permeability probably arose from modification of proteins of about 150–160 kDa, present in very
low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae).
HNE-induced promotion, via these proteins, of Ca2+ leakage pathways might be involved in the general toxic
effects of HNE.
channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives
to investigate the relationship between standard measures of hERG current block in an expression system and changes in
action potential duration (APD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The interference
from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model.
on the antioxidant activity of Thymbra capitata essential
oilwas made. This plant is a Mediterranean
culinary herb, whose essential oil antioxidant
power has recently been demonstrated in vitro as
one of the highest in nature. We tested if this in
vitro antioxidant capacity was reproducible on
biological systems using as model system primary
cultures of neonatal rat cardiomyocytes treated
with the lipid peroxidation product 4-hydroxy-2-
nonenal. The composition and the in vitro antioxidant
activity of the T. capitata essential oil were
also assessed. Cell viability, mitochondrial membrane
potential, and reactive oxygen species level
were measured in cells treated with pathophysiologic
doses of 4-hydroxy-2-nonenal (< 10 μM) or
vehicle after being pre-incubated with small concentrations
of the T. capitata essential oil, and the
ability of small doses (< 40 ppm) to prevent the
death of neonatal rat cardiomyocytes proved very
remarkable. Long-term pre-incubation (12 h)
with 20 ppm prevented 4-hydroxy-2-nonenal-induced
cell death and avoided mitochondrial
membrane potential loss and reactive oxygen species
generation caused by 4-hydroxy-2-nonenal.
A deleterious effect was shown at doses higher
than 40 ppm. The results of this study pave the
way to further analysis in animal models to
achieve a deeper understanding of the in vivo
antioxidant power of T. capitata essential oil.
maximal ATPase activity and Ca2+ transport ability of SERCA1a, the Ca2+ pump in these membranes. The
concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding
and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca2+ binding to
the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were
identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also
reacted with Lys515 within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the
steady-state ability of SR vesicles to sequester Ca2+ and the ATPase activity of SR membranes in the absence of
added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected
the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNEtreated
SR vesicles to Ca2+, an increase in permeability that did not arise from alteration of the lipid
component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent
increase in permeability probably arose from modification of proteins of about 150–160 kDa, present in very
low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae).
HNE-induced promotion, via these proteins, of Ca2+ leakage pathways might be involved in the general toxic
effects of HNE.