Antiapoptotic effect of nicorandil mediated by mitochondrial atp-sensitive potassium channels in cultured cardiac myocytes

AbstractObjectivesWe examined whether nicorandil, a clinically useful drug for the treatment of ischemic syndromes, inhibits myocardial apoptosis.BackgroundNicorandil has been reported to have a cardioprotective action through activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Based on our recent observation that mitoKATP channel activation has a remarkable antiapoptotic effect in cultured cardiac cells, we hypothesized that the protective effects of nicorandil may be at least partially due to an antiapoptotic effect.MethodsCultured neonatal rat cardiac myocytes were exposed to hydrogen peroxide to induce apoptosis. Effects of nicorandil were evaluated using a number of apoptotic markers.ResultsExposure to 100 μM hydrogen peroxide resulted in apoptotic cell death as shown by TUNEL positivity, cytochrome c translocation, caspase-3 activation and dissipation of mitochondrial inner membrane potential (ΔΨm). Nicorandil (100 μM) suppressed all of these markers of apoptosis. Notably, nicorandil prevented ΔΨm depolarization in a concentration-dependent manner (EC50 ∼ 40 μM, with saturation by 100 μM), as shown by fluorescence-activated cell sorter analysis of cells stained with a fluorescent ΔΨm-indicator, tetramethylrhodamine ethyl ester (TMRE). Time-lapse confocal microscopy of individual cells loaded with TMRE shows that nicorandil suppresses ΔΨm loss. Subcellular calcein localization revealed inhibition of the mitochondrial permeability transition by nicorandil. These protective effects of nicorandil were blocked by the mitoKATP channel antagonist 5-hydroxydecanoate.ConclusionsOur findings identify nicorandil as an inhibitor of apoptosis induced by oxidative stress in cardiac myocytes, and confirm the critical role of mitoKATP channels in inhibiting apoptosis

In vivo contrast free chronic myocardial infarction characterization using diffusion-weighted cardiovascular magnetic resonance.

BackgroundDespite the established role of late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) in characterizing chronic myocardial infarction (MI), a significant portion of chronic MI patients are contraindicative for the use of contrast agents. One promising alternative contrast free technique is diffusion weighted CMR (dwCMR), which has been shown ex vivo to be sensitive to myocardial fibrosis. We used a recently developed in vivo dwCMR in chronic MI pigs to compare apparent diffusion coefficient (ADC) maps with LGE imaging for infarct characterization.MethodsIn eleven mini pigs, chronic MI was induced by complete occlusion of the left anterior descending artery for 150 minutes. LGE, cine, and dwCMR imaging was performed 8 weeks post MI. ADC maps were derived from three orthogonal diffusion directions (b = 400 s/mm2) and one non-diffusion weighted image. Two semi-automatic infarct classification methods, threshold and full width half max (FWHM), were performed in both LGE and ADC maps. Regional wall motion (RWM) analysis was performed and compared to ADC maps to determine if any observed ADC change was significantly influenced by bulk motion.ResultsADC of chronic MI territories was significantly increased (threshold: 2.4 ± 0.3 μm2/ms, FWHM: 2.4 ± 0.2 μm2/ms) compared to remote myocardium (1.4 ± 0.3 μm2/ms). RWM was significantly reduced (threshold: 1.0 ± 0.4 mm, FWHM: 0.9 ± 0.4 mm) in infarcted regions delineated by ADC compared to remote myocardium (8.3 ± 0.1 mm). ADC-derived infarct volume and location had excellent agreement with LGE. Both LGE and ADC were in complete agreement when identifying transmural infarcts. Additionally, ADC was able to detect LGE-delineated infarcted segments with high sensitivity, specificity, PPV, and NPV. (threshold: 0.88, 0.93, 0.87, and 0.94, FWHM: 0.98, 0.97, 0.93, and 0.99, respectively).ConclusionsIn vivo diffusion weighted CMR has potential as a contrast free alternative for LGE in characterizing chronic MI

Charged Residues between the Selectivity Filter and S6 Segments Contribute to the Permeation Phenotype of the Sodium Channel

The deep regions of the Na+ channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd2+ block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd2+ binding to these residues reveal a secondary “dip” into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues

Dual gene therapy with SERCA1 and Kir2.1 abbreviates excitation without suppressing contractility

Heart failure is characterized by depressed contractility and delayed repolarization. The latter feature predisposes the failing heart to ventricular arrhythmias and represents a logical target for gene therapy. Unfortunately, unopposed correction of the delay in repolarization will decrease the time available for calcium cycling during each heartbeat, potentially aggravating the depression of contractility. Here we describe the development and application of a novel gene therapy strategy designed to abbreviate excitation without depressing contraction. The calcium ATPase SERCA1 was coexpressed with the potassium channel Kir2.1 in guinea pig hearts. Myocytes from the hearts had bigger calcium transients and shorter action potentials. In vivo, repolarization was abbreviated, but contractile function remained unimpaired. Dual gene therapy of the sort described here can be generalized to exploit opposing or synergistic therapeutic principles to achieve a tailored phenotype.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Dual gene therapy with SERCA1 and Kir2.1 abbreviates excitation without suppressing contractility

Heart failure is characterized by depressed contractility and delayed repolarization. The latter feature predisposes the failing heart to ventricular arrhythmias and represents a logical target for gene therapy. Unfortunately, unopposed correction of the delay in repolarization will decrease the time available for calcium cycling during each heartbeat, potentially aggravating the depression of contractility. Here we describe the development and application of a novel gene therapy strategy designed to abbreviate excitation without depressing contraction. The calcium ATPase SERCA1 was coexpressed with the potassium channel Kir2.1 in guinea pig hearts. Myocytes from the hearts had bigger calcium transients and shorter action potentials. In vivo, repolarization was abbreviated, but contractile function remained unimpaired. Dual gene therapy of the sort described here can be generalized to exploit opposing or synergistic therapeutic principles to achieve a tailored phenotype.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Molecular basis of isoform-specific μ-conotoxin block of cardiac, skeletal muscle, and brain Na+ channels

μ-Conotoxins (μ-CTXs) block skeletal muscle Na+ channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na+ channels. Although a number of conserved pore residues are recognized as critical determinants of μ-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (μ1, Nav1.4), human heart (hh1, Nav1.5), and rat brain (rb1, Nav1.1) Na+ channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Nav1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Nav1.1 and Nav1.5. The Nav1.4→Nav1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Nav1.4 numbering) reduced block of Nav1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Nav1.5→ Nav1.4 chimeric construct in which the Nav1.4 DII S5-P linker replaces the analogous segment in Nav1.5 showed enhanced μ-CTX block. However, these variant determinants are conserved between Nav1.1 and Nav1.4 and thus cannot explain their different sensitivities to μ-CTX. Comparison of their sequences reveals two variants at Nav1.4 positions 729 and 732: Ser and Asn in Nav1.4 compared with Thr and Lys in Nav1.1, respectively. The double mutation S729T/N732K rendered Nav1.4 more "brain-like" (30-fold ↓ in block), and the converse mutation T925S/K928N in Nav1.1 reproduced the high affinity blocking phenotype of Nav1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in μ-CTX binding, thus shaping isoform-specific toxin sensitivity.Facultad de Ciencias Médica

Viral gene transfer of dominant-negative Kv4 construct suppresses an O2-sensitive K+ current in chemoreceptor cells

Producción CientíficaHypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K+ channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K+ current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine + with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in poly- cistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O -sensitive K+ current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work dem- onstrate that genes of the Shal K+ channels underlie the tran- + their contribution to the native O2-sensitive K current. To probe sient outward, O2-sensitive, K current of rabbit CB chemore- the molecular identity of this current, we have used dominant- negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfam- ceptor cells and that this current contributes to the cell depolarization in response to low pO2. + ilies, because members of these two subfamilies contribute to Key words: O2-sensitive K current; viral gene transfer; the transient outward K+ currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved2018-03-2

Frequency-dependent changes in calcium cycling and contractile activation in SERCA2a transgenic mice

Objective: This study was undertaken to investigate the mechanism of altered contractility in hearts from transgenic mice overexpressing the sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a). In particular, we sought to determine whether the reported increase in contractility is freqnency-dependent, as might be expected if attributable to changes in SR Ca²⁺ loading. Methods: Intracellular [Ca²⁺] and contractile force were measured at room temperature (22 °C) simultaneously in fura-2-loaded isometrically-contracting trabeculae dissected from the hearts of FVB/N control (n=6) or SERCA2a transgenic (n=6) mice. Results: SERCA transgenics exhibit a positive force-frequency relationship, but this was flat in age- and strain-matched controls. SERCA transgenics exhibit a sizable increase in calcium transient amplitude relative to controls, with a concomitant increase in force generation at higher frequencies of stimulation. Amplitudes of Ca²⁺ transients (transgenics: 1.56 ± 0.09 μmol/l, controls: 1.21 ± 0.14) and twitches (transgenics: 21.71 ± 0.91 mN/mm², controls: 13.74 ± 1.67) were significantly different at 2.0 Hz stimulation (P < 0.05). Conclusion: An increase in SERCA expression increases the ability of the sarcoplasmic reticulum to store calcium, such that more calcium is available to be released during each heartbeat at higher stimulation rates.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare