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.cardiac tissue engineering ͉ myocardial infarction ͉ SDF-1 ͉ vascularization ͉ affinity-binding alginate scaffolds

MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from pro-inflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA-21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21 with a potential for use to attenuate post-MI remodeling and heart failure.

No abstract

(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 Ϯ 2.3% in rats (P Ͻ 0.0001; n ϭ 35), 31.7 Ϯ 8.3% in ICR mice (P ϭ 0.0001; n ϭ 13), and 57.7 Ϯ 13.7% in C57BL6 mice (P ϭ 0.015; n ϭ 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 remodelingreverse 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.atrial effective refractory period; rate adaptation; electrical remodeling MICE AND RATS ARE USED EXTENSIVELY in cardiac research, and reliable models of cardiac pathologies have been developed and applied in these species (4,19,31). Moreover, genetically altered mice have become invaluable tools for studying the molecular basis of cardiac pathologies including ventricular arrhythmias (7, 28). Use of rodents for studying atrial arrhythmias has been largely limited, but in recent years atrial tachyarrhythmias have been repeatedly produced in rats and mice, and rodents are increasingly used for studying various molecular, cellular, and pharmacological aspects of atrial function (1,5,8,24,36,37). In addition, mice with genetic alterations are providing important insights in this field as well (17,23,27,32).Due to the small size of the rodent heart and, more so, the rodent atria, electrode implantation for studies of functional electrophysiology is challenging. Several techniques were developed to increase the applicability of such studies (2, 23). The transesophageal approach, which is least invasive, necessitates the use of high e...

ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protecting cells against Zn(2+) toxicity. Our results demonstrate that ZnT-1 extrudes zinc from mammalian cells by functioning as a Zn(2+)/H(+) exchanger.

The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1-10 microM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca2+ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd2+, and low [Ca2+]o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 microM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells.

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory ␤-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the ␤-subunit. An interaction between ZnT-1 and the ␤-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming ␣ 1 -subunit of the LTCC. Similarly, a decrease in the surface expression of the ␣ 1 -subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC ␤-subunit and that it involves a decrease in the trafficking of the LTCC ␣ 1 -subunit to the surface membrane.

High pressure induces CNS hyperexcitability while markedly depressing synaptic transmitter release. We studied the effect of pressure (up to 10.1 MPa) on the parallel fibre (PF) synaptic response in biplanar cerebellar slices of adult guinea pigs. Pressure mildly reduced the PF volley amplitude and to a greater extent depressed the excitatory field postsynaptic potential (fPSP). The depression of the PF volley was noted even at supramaximal stimulus intensities, indicating an effect of pressure on the amplitude of the action potential in each axon. Low concentrations of TTX mimicked the effects of pressure on the PF volley without affecting the fPSP. Application omega-conotoxin GVIA (omega-CgTx) reduced the synaptic efficacy by 34.3+/-2.7%. However, in the presence of omega-CgTx the synaptic depression at pressure was significantly reduced. Reduced Ca2+ entry by application of Cd2+ or low [Ca2+]o did not have a similar influence on the effects of pressure. Application of omega-AGA IVA, omega-AGA TK and Funnel-web spider toxin did not affect the synaptic response in concentrations that usually block P-type Ca2+ channels, whilst the N/P/Q-type blocker omega-conotoxin MVIIC reduced the response to 52.7+/-5.0% indicating the involvement of Q-type channels and R-type channels in the non-N-type fraction of Ca2+ entry. The results demonstrate that N-type Ca2+ channels play a crucial role in the induction of PF synaptic depression at pressure. This finding suggests a coherent mechanism for the induction of CNS hyperexcitability at pressure.