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1.
Chaos ; 27(9): 093940, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28964110

RESUMEN

Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.


Asunto(s)
Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/fisiopatología , Calcio/metabolismo , Activación del Canal Iónico , Síndrome de QT Prolongado/complicaciones , Síndrome de QT Prolongado/fisiopatología , Modelos Cardiovasculares , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Potenciales de Acción , Animales , Simulación por Computador , Diástole , Conejos
2.
J Mol Cell Cardiol ; 81: 81-93, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25644533

RESUMEN

BACKGROUND AND OBJECTIVES: The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain. METHODS: Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions. RESULTS: The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1µM) or caffeine (100µM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2µM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2µM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules. CONCLUSIONS: Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.


Asunto(s)
Calcio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Compuestos de Anilina/farmacología , Animales , Benzazepinas/farmacología , Cafeína/farmacología , Fármacos Cardiovasculares/farmacología , Diferenciación Celular , Línea Celular , Reprogramación Celular , Conexina 43/metabolismo , Dependovirus/genética , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isoproterenol/farmacología , Ivabradina , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Éteres Fenílicos/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tetracaína/farmacología , Transfección
3.
Circ Res ; 113(3): 313-21, 2013 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-23748429

RESUMEN

RATIONALE: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy. OBJECTIVE: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca²âº transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P<0.01 versus WKY), steeper CV restitution kinetics, greater collagen deposition, higher levels of transcripts for transforming growth factor-ß, metalloproteinase-2, metalloproteinase-9, collagen I/III, and reduced connexin 43 phosphorylation (P<0.05 versus WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5) and SHR+V (n=4/4), but not in WKY (n=0/5) and SHR+RLX (n=1/8; P<0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV restitution kinetics, reduced action potential duration at 90% recovery to baseline, increased CV (P<0.01), and reversed atrial hypertrophy (P<0.05). Independent of antifibrotic actions, RLX (0.1 µmol/L) increased Na⁺ current density, INa (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P<0.01). CONCLUSIONS: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing INa. The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.


Asunto(s)
Fibrilación Atrial/tratamiento farmacológico , Cardiomiopatías/tratamiento farmacológico , Miocitos Cardíacos/fisiología , Relaxina/fisiología , Relaxina/uso terapéutico , Animales , Fibrilación Atrial/fisiopatología , Cardiomiopatías/fisiopatología , Fibrosis/fisiopatología , Fibrosis/prevención & control , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/fisiología , Humanos , Hipertrofia/tratamiento farmacológico , Hipertrofia/fisiopatología , Masculino , Miocitos Cardíacos/patología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico
4.
Am J Physiol Heart Circ Physiol ; 307(5): H640-8, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24973386

RESUMEN

Sexual dimorphism is a well-established phenomenon, but its degree varies tremendously among species. Since the early days of Einthoven's development of the three-lead galvanometer ECG, we have known there are marked differences in QT intervals of men and women. It required over a century to appreciate the profound implications of sex-based electrophysiological differences in QT interval on the panoply of sex differences with respect to arrhythmia risk, drug sensitivity, and treatment modalities. Little is known about the fundamental mechanism responsible for sex differences in electrical substrate of the human heart, in large part due to the lack of tissue availability. Animal models are an important research tool, but species differences in the sexual dimorphism of the QT interval, the ionic currents underlying the cardiac repolarization, and effects of sex steroids make it difficult to interpolate animal to human sex differences. In addition, in some species, different strains of the same animal model yield conflicting data. Each model has its strengths, such as ease of genetic manipulation in mice or size in dogs. However, many animals do not reproduce the sexual dimorphism of QT seen in humans. To match sex linked prolongation of QT interval and arrhythmogenic phenotype, the current data suggest that the rabbit may be best suited to provide insight into sex differences in humans. In the future, emerging technologies such as induced pluripotent stem cell derived cardiac myocyte systems may offer the opportunity to study sex differences in a controlled hormonal situation in the context of a sex specific human model system.


Asunto(s)
Potenciales de Acción , Hormonas Esteroides Gonadales/metabolismo , Síndrome de QT Prolongado/fisiopatología , Animales , Femenino , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Masculino , Factores Sexuales
5.
J Phys Chem A ; 118(42): 9837-43, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25216181

RESUMEN

The fluorescence of the SKC-513 ((E)-N-(9-(4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-6-(butyl(3-sulfopropyl)amino)-3H-xanthen-3-ylidene)-N-(3-sulfopropyl)butan-1-aminium) dye is shown experimentally to have high sensitivity to binding of the K(+) ion. Computations are used to explore the potential origins of this sensitivity and to make some suggestions regarding structural improvements. In the absence of K(+), excitation is to two nearly degenerate states, a neutral (N) excited state with a high oscillator strength, and a charge-transfer (CT) state with a lower oscillator strength. Binding of K(+) destabilizes the CT state, raising its energy far above the N state. The increase in fluorescence quantum yield upon binding of K(+) is attributed to the increased energy of the CT state suppressing a nonradiative pathway mediated by the CT state. The near degeneracy of the N and CT excited states can be understood by considering SKC-513 as a reduced symmetry version of a parent molecule with 3-fold symmetry. Computations show that acceptor-donor substituents can be used to alter the relative energies of the N and CT state, whereas a methylene spacer between the heterocycle and phenylene groups can be used to increase the coupling between these states. These modifications provide synthetic handles with which to optimize the dye for K(+) detection.


Asunto(s)
Colorantes Fluorescentes/química , Potasio/análisis , Simulación de Dinámica Molecular , Estructura Molecular , Teoría Cuántica , Espectrometría de Fluorescencia
6.
Biochem Pharmacol ; 227: 116407, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38969298

RESUMEN

Healthy aging results in cardiac structural and electrical remodeling that increase susceptibility to cardiovascular diseases. Relaxin has shown broad cardioprotective effects including anti-fibrotic, anti-arrhythmic and anti-inflammatory outcomes in multiple models. This paper focuses on the cardioprotective effects of Relaxin in a rat model of aging. Sustained atrial or ventricular fibrillation are readily induced in the hearts of aged but not young control animals. Treatment with Relaxin suppressed this arrhythmogenic response by increasing conduction velocity, decreasing fibrosis and promoting substantial cardiac remodeling. Relaxin treatment resulted in a significant increase in the levels of: Nav1.5, Cx43, ßcatenin and Wnt1 in rat hearts. In isolated cardiomyocytes, Relaxin increased Nav1.5 expression. These effects were mimicked by CHIR 99021, a pharmacological activator of canonical Wnt signaling, but blocked by the canonical Wnt inhibitor Dickkopf1. Relaxin prevented TGF-ß-dependent differentiation of cardiac fibroblasts into myofibroblasts while increasing the expression of Wnt1; the effects of Relaxin on cardiac fibroblast differentiation were blocked by Dickkopf1. RNASeq studies demonstrated reduced expression of pro-inflammatory cytokines and an increase in the expression of α- and ß-globin in Relaxin-treated aged males. Relaxin reduces arrhythmogenicity in the hearts of aged rats by reduction of fibrosis and increased conduction velocity. These changes are accompanied by substantial remodeling of the cardiac tissue and appear to be mediated by increased canonical Wnt signaling. Relaxin also exerts significant anti-inflammatory and anti-oxidant effects in the hearts of aged rodents. The mechanisms by which Relaxin increases the expression of Wnt ligands, promotes Wnt signaling and reprograms gene expression remain to be determined.


Asunto(s)
Envejecimiento , Fibrilación Atrial , Fibrosis , Relaxina , Animales , Relaxina/farmacología , Masculino , Ratas , Fibrilación Atrial/metabolismo , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/prevención & control , Envejecimiento/efectos de los fármacos , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Ratas Endogámicas F344
7.
Biochem Pharmacol ; 223: 116136, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38494063

RESUMEN

Relaxin-2 (RLX), a critical hormone in pregnancy, has been investigated as a therapy for heart failure. In most studies, the peptide was delivered continuously, subcutaneously for 2 weeks in animals or intravenously for 2-days in human subjects, for stable circulating [RLX]. However, pulsatile hormone levels may better uncover the normal physiology. This premise was tested by subcutaneously injecting Sprague Dawley rats (250 g, N = 2 males, 2 females/group) with human RLX (0, 30, 100, or 500 µg/kg), every 12 h for 1 day, then measuring changes in Nav1.5, connexin43, and ß-catenin, 24 h later. Pulsatile RLX was measured by taking serial blood draws, post-injection. After an injection, RLX reached a peak in âˆ¼ 60 min, fell to 50 % in 5-6 h; injections of 0, 30, 100 or 500 µg/kg yielded peak levels of 0, 11.26 ± 3.52, 58.33 ± 16.10, and 209.42 ± 29.04 ng/ml and residual levels after 24-hrs of 0, 4.9, 45.1 and 156 pg/ml, respectively. The 30 µg/kg injections had no effect and 100 µg/kg injections increased Nav1.5 (25 %), Cx43 (30 %) and ß-catenin (90 %). The 500 µg/kg injections also increased Nav1.5 and Cx43 but were less effective at upregulating ß-catenin (up by 25 % vs. 90 %). Periodic injections of 100 µg/kg were highly effective at increasing the expression of Nav1.5 and Cx43 which are key determinants of conduction velocity in the heart and the suppression of arrhythmias. Periodic RLX is effective at eliciting changes in cardiac protein expression and may be a better strategy for its longer-term delivery in the clinical setting.


Asunto(s)
Relaxina , Embarazo , Ratas , Masculino , Animales , Femenino , Humanos , Relaxina/metabolismo , beta Catenina , Conexina 43/genética , Ratas Sprague-Dawley , Arritmias Cardíacas
8.
Am J Physiol Heart Circ Physiol ; 304(6): H848-60, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23316064

RESUMEN

Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca(2+) transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 µm(2)) or low (1.5 × 1.5 cm(2)) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant τf→s = 48 ± 9.2 s) and caused a secondary Ca(2+) release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca(2+) transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca(2+) release events leading to subcellular Ca(2+) waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 µM), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca(2+) channel protein (Cav1.2α), Na(+)-Ca(2+) exchange (NCX1), voltage-gated Na(+) channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca(2+) ATPase 2a] that correlate with the SCR distribution and explain the molecular basis for SCR heterogeneities. In conclusion, acute bradycardia elicits synchronized subcellular SCRs of sufficient magnitude to overcome the source-sink mismatch and to promote afterdepolarizations.


Asunto(s)
Potenciales de Acción , Bradicardia/metabolismo , Señalización del Calcio/fisiología , Taquicardia/etiología , Animales , Bradicardia/fisiopatología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Regulación de la Expresión Génica , Frecuencia Cardíaca , Técnicas In Vitro , Reperfusión Miocárdica , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismo , Tiazepinas/farmacología
9.
Nature ; 450(7171): 819-24, 2007 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-18064002

RESUMEN

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.


Asunto(s)
Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/prevención & control , Conexina 43/metabolismo , Infarto del Miocardio/complicaciones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Animales , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Conexina 43/genética , Embrión de Mamíferos/citología , Corazón/fisiología , Corazón/fisiopatología , Humanos , Ratones , Ratones Transgénicos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/citología , Miocardio/patología , Perfusión
10.
Front Cardiovasc Med ; 10: 1158480, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37153461

RESUMEN

Background and significance: The specialized conduction system (SCS) of the heart was extensively studied to understand the synchronization of atrial and ventricular contractions, the large atrial to His bundle (A-H) delay through the atrioventricular node (AVN), and delays between Purkinje (P) and ventricular (V) depolarization at distinct junctions (J), PVJs. Here, we use optical mapping of perfused rabbit hearts to revisit the mechanism that explains A-H delay and the role of a passive electrotonic step-delay at the boundary between atria and the AVN. We further visualize how the P anatomy controls papillary activation and valve closure before ventricular activation. Methods: Rabbit hearts were perfused with a bolus (100-200 µl) of a voltage-sensitive dye (di4ANEPPS), blebbistatin (10-20 µM for 20 min) then the right atrial appendage and ventricular free-wall were cut to expose the AVN, P fibers (PFs), the septum, papillary muscles, and the endocardium. Fluorescence images were focused on a CMOS camera (SciMedia) captured at 1K-5 K frames/s from 100 × 100 pixels. Results: AP propagation across the AVN-His (A-H) exhibits distinct patterns of delay and conduction blocks during S1-S2 stimulation. Refractory periods were 81 ± 9, 90 ± 21, 185 ± 15 ms for Atrial, AVN, and His, respectively. A large delay (>40 ms) occurs between atrial and AVN activation that increased during rapid atrial pacing contributing to the development of Wenckebach periodicity followed by delays within the AVN through slow or blocked conduction. The temporal resolution of the camera allowed us to identify PVJs by detecting doublets of AP upstrokes. PVJ delays were heterogeneous, fastest in PVJ that immediately trigger ventricular APs (3.4 ± 0.8 ms) and slow in regions where PF appear insulated from the neighboring ventricular myocytes (7.8 ± 2.4 ms). Insulated PF along papillary muscles conducted APs (>2 m/s), then triggered papillary muscle APs (<1 m/s), followed by APs firing of septum and endocardium. The anatomy of PFs and PVJs produced activation patterns that control the sequence of contractions ensuring that papillary contractions close the tricuspid valve 2-5 ms before right ventricular contractions. Conclusions: The specialized conduction system can be accessed optically to investigate the electrical properties of the AVN, PVJ and activation patterns in physiological and pathological conditions.

11.
Front Chem ; 11: 1220573, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37547910

RESUMEN

Modern medicine has developed a myriad of therapeutic drugs against a wide range of human diseases leading to increased life expectancy and better quality of life for millions of people. Despite the undeniable benefit of medical advancements in pharmaceutical technology, many of the most effective drugs currently in use have serious limitations such as off target side effects resulting in systemic toxicity. New generations of specialized drug constructs will enhance targeted therapeutic efficacy of existing and new drugs leading to safer and more effective treatment options for a variety of human ailments. As one of the most efficient drugs known for the treatment of cardiac arrhythmia, Amiodarone presents the same conundrum of serious systemic side effects associated with long term treatment. In this article we present the synthesis of a next-generation prodrug construct of amiodarone for the purpose of advanced targeting of cardiac arrhythmias by delivering the drug to cardiomyocytes using a novel cardiac targeting peptide, a cardiomyocyte-specific cell penetrating peptide. Our in vivo studies in guinea pigs indicate that cardiac targeting peptide-amiodarone conjugate is able to have similar effects on calcium handling as amiodarone at 1/15th the total molar dose of amiodarone. Further studies are warranted in animal models of atrial fibrillation to show efficacy of this conjugate.

12.
Pharmaceutics ; 15(8)2023 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-37631321

RESUMEN

BACKGROUND: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. METHODS: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP-amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570-595 nm for the cytosolic Ca2+ and 610-750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. RESULTS: The CTP-amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP-amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP-amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP-amiodarone significantly decreased the calcium transients compared to the controls. CTP-amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

13.
bioRxiv ; 2023 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-37214919

RESUMEN

Background: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to lowering of dose by utilizing a cardiomyocyte targeting peptide (CTP), a cell penetrating peptide identified by our prior phage display work. Methods: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified and confirmed with MALDI/TOF. Stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), CTP-amiodarone (5 days;26.3mg/Kg), or CTP (5 days; 17.8mg/Kg), after which GPs were euthanized, hearts excised, perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5µM) to minimize contractions. Voltage (RH237) and Ca 2+ -indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca 2+ and 610-750nm wavelengths for voltage. Subsequently, hearts were paced at 250ms with programmed stimulation to measure changes in conduction velocities (CV), action potential duration (APD) and Ca 2+ transient durations at 90% recovery (CaTD 90 ). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts. Results: CTP-amiodarone remained stable for up to 21 days at 37°C. At ∼1/15 th of the dose of amiodarone, CTP-amiodarone decreased CV in hearts significantly compared to control GPs (0.92±0.05 vs. 1.00±0.03m/s, p=0.0007), equivalent to amiodarone alone (0.87±0.08ms, p=0.0003). Amiodarone increased APD (192±5ms vs. 175±8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157±16ms, p=0.0136) similar to CTP alone (155±13ms, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased calcium transients compared to controls. CTP-amiodarone and CTP decreased CaTD 90 to an extent greater than amiodarone alone (p<0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while decreasing expression of proinflammatory genes NF-kappa B, TNF-α, IL-1ß, and IL-6. Conclusions: Our data suggests that CTP can deliver amiodarone to cardiomyocytes at ∼1/15 th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD 90 may be related to its increase in expression of Ca-handling genes, and merits further study.

14.
J Clin Transl Sci ; 7(1): e215, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37900348

RESUMEN

The National Institutes of Health's (NIH) K99/R00 Pathway to Independence Award offers promising postdoctoral researchers and clinician-scientists an opportunity to receive research support at both the mentored and the independent levels with the goal of facilitating a timely transition to a tenure-track faculty position. This transitional program has been generally successful, with most K99/R00 awardees successfully securing R01-equivalent funding by the end of the R00 period. However, often highly promising proposals fail because of poor grantsmanship. This overview provides guidance from the perspective of long-standing members of the National Heart, Lung, and Blood Institute's Mentored Transition to Independence study section for the purpose of helping mentors and trainees regarding how best to assemble competitive K99/R00 applications.

15.
J Physiol ; 590(3): 493-508, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22124151

RESUMEN

In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels of L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I(Ca,L) density is upregulated in adult female rabbit hearts. I(Ca,L) density was measured by the whole-cell patch-clamp technique on days 0-3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I(Ca,L) was 28% higher at the base than apex (P < 0.01) and decreased gradually (days 0-3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1-1.0 nm) increased I(Ca,L) (∼2-fold) in female base but not endo-, apex or male myocytes. Progesterone (0.1-10 µm) had no effect at base myocytes. An agonist of the α- (PPT, 5 nm) but not the ß- (DPN, 5 nm) subtype oestrogen receptor (ERα/ERß) upregulated I(Ca,L) like E2. Western blots detected similar levels of ERα and ERß in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT-PCR) levels but did not change I(Ca,L) kinetics. I(Ca,L) upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 µm) or by inhibition of transcription (actinomycin D, 4 µm) or protein biosynthesis (cycloheximide, 70 µm). Therefore, E2 upregulates I(Ca,L) by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.


Asunto(s)
Canales de Calcio Tipo L/fisiología , Estradiol/farmacología , Receptor alfa de Estrógeno/fisiología , Estrógenos/farmacología , Corazón/fisiología , Animales , Células Cultivadas , Femenino , Masculino , Células Musculares/efectos de los fármacos , Células Musculares/fisiología , Progesterona/farmacología , Progestinas/farmacología , ARN Mensajero/metabolismo , Conejos , Caracteres Sexuales
16.
Am J Physiol Heart Circ Physiol ; 302(4): H934-52, 2012 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-22081697

RESUMEN

Transgenic mice overexpressing tumor necrosis factor-α (TNF-α mice) possess many of the features of human heart failure, such as dilated cardiomyopathy, impaired Ca(2+) handling, arrhythmias, and decreased survival. Although TNF-α mice have been studied extensively with a number of experimental methods, the mechanisms of heart failure are not completely understood. We created a mathematical model that reproduced experimentally observed changes in the action potential (AP) and Ca(2+) handling of isolated TNF-α mice ventricular myocytes. To study the contribution of the differences in ion currents, AP, Ca(2+) handling, and intercellular coupling to the development of arrhythmias in TNF-α mice, we further created several multicellular model tissues with combinations of wild-type (WT)/reduced gap junction conductance, WT/prolonged AP, and WT/decreased Na(+) current (I(Na)) amplitude. All model tissues were examined for susceptibility to Ca(2+) alternans, AP propagation block, and reentry. Our modeling results demonstrated that, similar to experimental data in TNF-α mice, Ca(2+) alternans in TNF-α tissues developed at longer basic cycle lengths. The greater susceptibility to Ca(2+) alternans was attributed to the prolonged AP, resulting in larger inactivation of I(Na), and to the decreased SR Ca(2+) uptake and corresponding smaller SR Ca(2+) load. Simulations demonstrated that AP prolongation induces an increased susceptibility to AP propagation block. Programmed stimulation of the model tissues with a premature impulse showed that reduced gap junction conduction increased the vulnerable window for initiation reentry, supporting the idea that reduced intercellular coupling is the major factor for reentrant arrhythmias in TNF-α mice.


Asunto(s)
Arritmias Cardíacas/metabolismo , Modelos Teóricos , Miocardio/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/fisiología , Potenciales de Acción/fisiología , Animales , Arritmias Cardíacas/fisiopatología , Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Uniones Comunicantes/fisiología , Ratones , Ratones Transgénicos , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba/genética
17.
Nat Med ; 11(4): 409-17, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15793582

RESUMEN

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.


Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Antagonistas Adrenérgicos beta/farmacología , Animales , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Gasto Cardíaco Bajo , Cardiomegalia , Ratones , Ratones Transgénicos , Contracción Miocárdica , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Fosforilación , Remodelación Ventricular
18.
JCI Insight ; 7(3)2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35132963

RESUMEN

Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.


Asunto(s)
Mapeo del Potencial de Superficie Corporal/métodos , Infarto del Miocardio/diagnóstico , Miocardio/patología , Sistema Nervioso Simpático/diagnóstico por imagen , Potenciales de Acción , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/fisiopatología , Sistema Nervioso Simpático/fisiopatología
19.
Cardiovasc Res ; 118(9): 2124-2138, 2022 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-34329394

RESUMEN

AIMS: The cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. METHODS AND RESULTS: We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP-deficient (Nppa-/-) or BNP-deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human-derived induced pluripotent stem cell cardiomyocytes (iPS-CMs). In the unstressed state, both ANP- and BNP-deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodelling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress-induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild-type hearts to stress-induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen-activated protein kinase (p38 MAPK) signalling cascade. CONCLUSIONS: Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signalling which sensitizes the heart to stress-induced ventricular arrhythmias.


Asunto(s)
Arritmias Cardíacas , Factor Natriurético Atrial , Péptido Natriurético Encefálico , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , GMP Cíclico , Ratones , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Péptidos Natriuréticos/metabolismo , Vasodilatadores , Proteínas Quinasas p38 Activadas por Mitógenos
20.
J Clin Invest ; 132(18)2022 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-36106636

RESUMEN

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.


Asunto(s)
Insuficiencia Cardíaca , Miocitos Cardíacos , Animales , Muerte Súbita Cardíaca , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Volumen Sistólico
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