Quantifying Intermembrane Distances with Serial Image Dilations

Raisch, Tristan B.; Khan, Momina; Poelzing, Steven

doi:10.3791/58311

Cited by 9 publications

(7 citation statements)

References 26 publications

(42 reference statements)

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“…In contrast, the addition of dextran 70 does not significantly alter w p relative to control. For comparison, we reanalyze our previously collected images from mannitolperfused hearts with the updated w p measurement algorithm, and the results support our previous finding that mannitol significantly increases w p compared with control (25,44). Importantly, w p measurements in hearts with the osmotic agents in the presence of ATXII are comparable to our previous measurements in the presence of the osmotic agents alone (24,25,37).…”

Section: Ex Vivosupporting

confidence: 72%

“…We first investigate a drug-induced Na þ GOF guinea pig model by adding three osmotic agents to modulate extracellular spaces. As in our previous work (25), TEM image analysis with an updated semiautomated measurement algorithm (44) shows that agent ATXII, inducing late Na þ current, does not alter width in the GJ-adjacent perinexal region ðw p Þ, compared with control (Fig. 2).…”

Section: Ex Vivosupporting

confidence: 71%

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Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

Nowak

Greer-Short

Wan

et al. 2020

Biophysical Journal

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In cardiac myocytes, action potentials are initiated by an influx of sodium (Na þ ) ions via voltage-gated Na þ channels. Na þ channel gain of function (GOF), arising in both inherited conditions associated with mutation in the gene encoding the Na þ channel and acquired conditions associated with heart failure, ischemia, and atrial fibrillation, enhance Na þ influx, generating a late Na þ current that prolongs action potential duration (APD) and triggering proarrhythmic early afterdepolarizations (EADs). Recent studies have shown that Na þ channels are highly clustered at the myocyte intercalated disk, facilitating formation of Na þ nanodomains in the intercellular cleft between cells. Simulations from our group have recently predicted that narrowing the width of the intercellular cleft can suppress APD prolongation and EADs in the presence of Na þ channel mutations because of increased intercellular cleft Na þ ion depletion. In this study, we investigate the effects of modulating multiple extracellular spaces, specifically the intercellular cleft and bulk interstitial space, in a novel computational model and experimentally via osmotic agents albumin, dextran 70, and mannitol. We perform optical mapping and transmission electron microscopy in a drug-induced (sea anemone toxin, ATXII) Na þ channel GOF isolated heart model and modulate extracellular spaces via osmotic agents. Singlecell patch-clamp experiments confirmed that the osmotic agents individually do not enhance late Na þ current. Both experiments and simulations are consistent with the conclusion that intercellular cleft narrowing or expansion regulates APD prolongation; in contrast, modulating the bulk interstitial space has negligible effects on repolarization. Thus, we predict that intercellular cleft Na þ nanodomain formation and collapse critically regulates cardiac repolarization in the setting of Na þ channel GOF.

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Section: Ex Vivosupporting

confidence: 72%

Section: Ex Vivosupporting

confidence: 71%

Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

Nowak

Greer-Short

Wan

et al. 2020

Biophysical Journal

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“…However, VEGF's effects on GJs have been demonstrate to occur over much longer time courses (> 4 h) than those involved in the present study (< 4 h). Intermembrane spacing measured by TEM may have been impacted the effects of glutaraldehyde fixation on tissue 63 . However, such effects would uniformly impact all samples and do not detract from the observation that VEGF increases intermembrane spacing near GJs and MJs.…”

Section: Discussionmentioning

confidence: 99%

Vascular endothelial growth factor promotes atrial arrhythmias by inducing acute intercalated disk remodeling

Mezache

Struckman

Greer-Short

et al. 2020

Sci Rep

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Atrial fibrillation (AF) is the most common arrhythmia and is associated with inflammation. AF patients have elevated levels of inflammatory cytokines known to promote vascular leak, such as vascular endothelial growth factor A (VEGF). However, the contribution of vascular leak and consequent cardiac edema to the genesis of atrial arrhythmias remains unknown. Previous work suggests that interstitial edema in the heart can acutely promote ventricular arrhythmias by disrupting ventricular myocyte intercalated disk (ID) nanodomains rich in cardiac sodium channels (NaV1.5) and slowing cardiac conduction. Interestingly, similar disruption of ID nanodomains has been identified in atrial samples from AF patients. Therefore, we tested the hypothesis that VEGF-induced vascular leak can acutely increase atrial arrhythmia susceptibility by disrupting ID nanodomains and slowing atrial conduction. Treatment of murine hearts with VEGF (30–60 min, at clinically relevant levels) prolonged the electrocardiographic P wave and increased susceptibility to burst pacing-induced atrial arrhythmias. Optical voltage mapping revealed slower atrial conduction following VEGF treatment (10 ± 0.4 cm/s vs. 21 ± 1 cm/s at baseline, p < 0.05). Transmission electron microscopy revealed increased intermembrane spacing at ID sites adjacent to gap junctions (GJs; 64 ± 9 nm versus 17 ± 1 nm in controls, p < 0.05), as well as sites next to mechanical junctions (MJs; 63 ± 4 nm versus 27 ± 2 nm in controls, p < 0.05) in VEGF–treated hearts relative to controls. Importantly, super-resolution microscopy and quantitative image analysis revealed reorganization of NaV1.5 away from dense clusters localized near GJs and MJs to a more diffuse distribution throughout the ID. Taken together, these data suggest that VEGF can acutely predispose otherwise normal hearts to atrial arrhythmias by dynamically disrupting NaV1.5-rich ID nanodomains and slowing atrial conduction. These data highlight inflammation-induced vascular leak as a potential factor in the development and progression of AF.

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“…Images (average of 16 per heart) of perinexi and their associated GJs within the ID were obtained using a JEOL JEM1400 electron microscope at 150,000ϫ magnification. Perinexal width (W p) was measured within the range of 30 -105 nm from the edge of the GJ plaque using ImageJ and a MatLab-based algorithm recently developed by our group (65).…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

Attenuating loss of cardiac conduction during no-flow ischemia through changes in perfusate sodium and calcium

Hoeker

James

Tegge

et al. 2020

American Journal of Physiology-Heart and Circulatory Physiology

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Myocardial ischemia leads to conduction slowing, cell-to-cell uncoupling, and arrhythmias. We previously demonstrated that varying perfusate sodium (Na+) and calcium (Ca2+) attenuates conduction slowing and arrhythmias during simulated ischemia with continuous perfusion. Cardioprotection was selectively associated with widening of the perinexus, a gap junction adjacent nanodomain important to ephaptic coupling. It is unknown whether perfusate composition affects the perinexus or ischemic conduction during non-simulated ischemia, when coronary flow is reduced or halted. We hypothesized that altering pre-ischemic perfusate composition could facilitate perinexal expansion and attenuate conduction slowing during global ischemia. To test this hypothesis, ex vivo guinea pig hearts (n=49) were Langendorff-perfused with 145 or 153mM Na+ and 1.25 or 2.0mM Ca2+, and optically mapped during 30 minutes of no-flow ischemia. Altering Na+ and Ca2+ did not significantly affect baseline conduction velocity (CV). Increasing Na+ and decreasing Ca2+ both lowered pacing thresholds, while increasing Ca2+ narrowed perinexal width (WP). A least squared means estimate revealed that reduced Na+ and Ca2+ slowed CV by 2.20 (cm/s)/min of ischemia. Increasing Ca2+ significantly attenuated CV slowing to 1.51 (cm/s)/min. Increasing Na+ attenuated CV slowing to 0.99 (cm/s)/min, and delayed median time to conduction block (10 to 16 minutes). Increasing Na+ and Ca2+ selectively widened WP during ischemia (22.7 vs 15.7nm) and attenuated CV slowing to the greatest extent (0.66 (cm/s)/min). Neither repolarization nor levels of total or phosphorylated connexin43 correlated with conduction slowing or block. Thus, perfusate-dependent widening of the perinexus preserved ischemic conduction and may be an adaptive response to ischemic stress.

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Quantifying Intermembrane Distances with Serial Image Dilations

Cited by 9 publications

References 26 publications

Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

Vascular endothelial growth factor promotes atrial arrhythmias by inducing acute intercalated disk remodeling

Attenuating loss of cardiac conduction during no-flow ischemia through changes in perfusate sodium and calcium

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