Shani Dror

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling

Activation of ERK signaling may promote cardioprotection from ischemia–reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

New insights into the atrial electrophysiology of rodents using a novel modality: the miniature-bipolar hook electrode

Studies of atrial electrophysiology (EP) in rodents are challenging, and available data are sparse. Herein, we utilized a novel type of bipolar electrode to evaluate the atrial EP of rodents through small lateral thoracotomy. In anesthetized rats and mice, we attached two bipolar electrodes to the right atrium and a third to the right ventricle. This standard setup enabled high-resolution EP studies. Moreover, a permanent implantation procedure enabled EP studies in conscious freely moving rats. Atrial EP was evaluated in anesthetized rats, anesthetized mice (ICR and C57BL6 strains), and conscious rats. Signal resolution enabled atrial effective refractory period (AERP) measurements and first time evaluation of the failed 1:1 atrial capture, which was unexpectedly longer than the AERP recorded at near normal cycle length by 27.2+/-2.3% in rats (P<0.0001; n=35), 31.7+/-8.3% in ICR mice (P=0.0001; n=13), and 57.7+/-13.7% in C57BL6 mice (P=0.015; n=4). While AERP rate adaptation was noted when 10 S1s at near normal basic cycle lengths were followed by S2 at varying basic cycle length and S3 for AERP evaluation, such rate adaptation was absent using conventional S1S2 protocols. Atrial tachypacing in rats shortened the AERP values on a timescale of hours, but a reverse remodeling phase was noted thereafter. Comparison of left vs. right atrial pacing in rats was also feasible with the current technique, resulting in similar AERP values recorded in the low right atrium. In conclusion, our findings indicate that in vivo rate adaptation of the rodent atria is different than expected based on previous ex vivo recordings. In addition, atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time. Further understanding of these properties should help to determine the clinical relevance as well as limitations of atrial arrhythmia models in rodents.

INO-8875, a Highly Selective A1 Adenosine Receptor Agonist: Evaluation of Chronotropic, Dromotropic, and Hemodynamic Effects in Rats

Selective pharmacological activation of the adenosine 1 receptor (A1R) is a promising new approach to achieve a potent block of atrioventricular (A-V)–nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A1R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V–nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 μM) progressively prolonged A-V–nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 μg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1–50 μg/kg) and CVT-510 (20 and 50 μg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V–nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V–nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A1R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.

Speckle-tracking echocardiography elucidates the effect of pacing site on left ventricular synchronization in the normal and infarcted rat myocardium.

Background Right ventricular (RV) pacing generates regional disparities in electrical activation and mechanical function (ventricular dyssynchrony). In contrast, left ventricular (LV) or biventricular (BIV) pacing can improve cardiac efficiency in the setting of ventricular dyssynchrony, constituting the rationale for cardiac resynchronization therapy (CRT). Animal models of ventricular dyssynchrony and CRT currently relay on large mammals which are expensive and not readily available to most researchers. We developed a methodology for double-site epicardial pacing in conscious rats. Here, following post-operative recovery, we compared the effects of various pacing modes on LV dyssynchrony in normal rats and in rats with ischemic cardiomyopathy. Methods Two bipolar electrodes were implanted in rats as follows: Group A (n = 6) right atrial (RA) and RV sites; Group B (n = 7) RV and LV sites; Group C (n = 8) as in group B in combination with left coronary artery ligation. Electrodes were exteriorized through the back. Following post-operative recovery, two-dimensional transthoracic echocardiography was performed during pacing through the different electrodes. Segmental systolic circumferential strain (Ecc) was used to evaluate LV dyssynchrony. Results In normal rats, RV pacing induced marked LV dyssynchrony compared to RA pacing or sinus rhythm, as measured by the standard deviation (SD) of segmental time to peak Ecc, SD of peak Ecc, and the average delay between opposing ventricular segments. LV pacing and, to a greater extend BIV pacing diminished the LV dyssynchrony compared to RV pacing. In rats with extensive MI, the effects of LV and BIV pacing were markedly attenuated, and the response of individual animals was variable. Conclusions Rodent cardiac pacing mimics important features seen in humans. This model may be developed as a simple new tool to study the pathophysiology of ventricular dyssynchrony and CRT.

The involvement of ZnT-1, a new modulator of cardiac L-type calcium channels, in remodeling atrial tachycardia

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self‐perpetuating nature of AF, is still unknown. Downregulation of L‐type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT‐1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT‐1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT‐1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT‐1 expression was found to be augmented in the atria of AF patients. These findings position ZnT‐1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.

The involvement of ZnT-1, a new modulator of cardiac L-type calcium channels, in [corrected] atrial tachycardia remodeling. [corrected].

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self‐perpetuating nature of AF, is still unknown. Downregulation of L‐type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT‐1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT‐1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT‐1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT‐1 expression was found to be augmented in the atria of AF patients. These findings position ZnT‐1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.

The involvement of ZnT-1, a new modulator of cardiac L-type calcium channels, in remodeling atrial tachycardia: ZnT-1, calcium channels, and atrial electrical remodeling

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self‐perpetuating nature of AF, is still unknown. Downregulation of L‐type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT‐1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT‐1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT‐1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT‐1 expression was found to be augmented in the atria of AF patients. These findings position ZnT‐1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.