Spatial Pattern Analysis using Closest Events (SPACE)-A Nearest Neighbor Point Pattern Analysis Framework for Assessing Spatial Relationships from Digital Images.

The quantitative description of biological structures is a valuable yet difficult task in the life sciences. This is commonly accomplished by imaging samples using fluorescence microscopy and analyzing resulting images using Pearson's correlation or Manders' co-occurrence intensity-based colocalization paradigms. Though conceptually and computationally simple, these approaches are critically flawed due to their reliance on signal overlap, sensitivity to cursory signal qualities, and inability to differentiate true and incidental colocalization. Point pattern analysis provides a framework for quantitative characterization of spatial relationships between spatial patterns using the distances between observations rather than their overlap, thus overcoming these issues. Here we introduce an image analysis tool called Spatial Pattern Analysis using Closest Events (SPACE) that leverages nearest neighbor-based point pattern analysis to characterize the spatial relationship of fluorescence microscopy signals from image data. The utility of SPACE is demonstrated by assessing the spatial association between mRNA and cell nuclei from confocal images of cardiac myocytes. Additionally, we use synthetic and empirical images to characterize the sensitivity of SPACE to image segmentation parameters and cursory image qualities such as signal abundance and image resolution. Ultimately, SPACE delivers performance superior to traditional colocalization methods and offers a valuable addition to the microscopist's toolbox.

Cardiac-Specific Deletion of Scn8a Mitigates Dravet Syndrome-Associated Sudden Death in Adults.

BACKGROUND: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear. OBJECTIVES: In this study, the authors sought to provide evidence implicating remodeling of Na+ - and Ca2+ -handling machinery, including NaV 1.6 and Na+/Ca2+exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias. METHODS: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS. RESULTS: DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death. CONCLUSIONS: These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Unraveling Impacts of Chamber-Specific Differences in Intercalated Disc Ultrastructure and Molecular Organization on Cardiac Conduction.

BACKGROUND: Propagation of action potentials through the heart coordinates the heartbeat. Thus, intercalated discs, specialized cell-cell contact sites that provide electrical and mechanical coupling between cardiomyocytes, are an important target for study. Impaired propagation leads to arrhythmias in many pathologies, where intercalated disc remodeling is a common finding, hence the importance and urgency of understanding propagation dependence on intercalated disc structure. Conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, because of lack of quantitative structural data at subcellular through nano scales. OBJECTIVES: This study sought to quantify intercalated disc structure at these spatial scales in the healthy adult mouse heart and relate them to chamber-specific properties of propagation as a precursor to understanding the effects of pathological intercalated disc remodeling. METHODS: Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. RESULTS: By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by interchamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. CONCLUSIONS: These data provide the first stepping stone to elucidating chamber-specific effects of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.

Unraveling Chamber-specific Differences in Intercalated Disc Ultrastructure and Molecular Organization and Their Impact on Cardiac Conduction.

During each heartbeat, the propagation of action potentials through the heart coordinates the contraction of billions of individual cardiomyocytes and is thus, a critical life process. Unsurprisingly, intercalated discs, which are cell-cell contact sites specialized to provide electrical and mechanical coupling between adjacent cardiomyocytes, have been the focus of much investigation. Slowed or disrupted propagation leads to potentially life-threatening arrhythmias in a wide range of pathologies, where intercalated disc remodeling is a common finding. Hence, the importance and urgency of understanding intercalated disc structure and its influence on action potential propagation. Surprisingly, however, conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, owing to lack of quantitative structural data at subcellular through nano scales. In order to address this critical gap in knowledge, we sought to quantify intercalated disc structure at these finer spatial scales in the healthy adult mouse heart and relate them to function in a chamber-specific manner as a precursor to understanding the impacts of pathological intercalated disc remodeling. Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by inter-chamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. These data provide the first stepping stone to elucidating chamber-specific impacts of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Tunable alginate hydrogels as injectable drug delivery vehicles for optic neuropathy.

Many disease pathologies, particularly in the eye, are induced by oxidative stress. In particular, injury to the optic nerve (ON), or optic neuropathy, is one of the most common causes of vision loss. Traumatic optic neuropathy (TON) occurs when the ON is damaged following blunt or penetrating trauma to either the head or eye. Currently, there is no effective treatment for TON, only management options, namely the systematic delivery of corticosteroids and surgical decompression of the optic nerve. Unfortunately, neither option alleviates the generation of reactive oxygen species (ROS) which are responsible for downstream damage to the ON. Additionally, the systemic delivery of corticosteroids can cause fatal off-target effects in cases with brain involvement. In this study, we developed a tunable injectable hydrogel delivery system for local methylene blue (MB) delivery using an internal method of crosslinking. MB was chosen due to its ROS scavenging ability and neuroprotective properties. Our MB-loaded polymeric scaffold demonstrated prolonged release of MB as well as in situ gel formation. Additionally, following rheological characterization, these alginate hydrogels demonstrated minimal cytotoxicity to human retinal pigment epithelial cells in vitro and exhibited injection feasibility through small-gauge needles. Our chosen MB concentrations displayed a high degree of ROS scavenging following release from the alginate hydrogels, suggesting this approach may be successful in reducing ROS levels following ON injury, or could be applied to other ocular injuries.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes.

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease.

Automated Gait Analysis Detects Improvements after Intracellular σ Peptide Administration in a Rat Hemisection Model of Spinal Cord Injury.

A promising treatment strategy for spinal cord injury (SCI) is to reduce inhibition from chondroitin sulfate proteoglycans (CSPGs). For example, administering intracellular σ peptide (ISP) can improve the ability of axons to cross inhibitory CSPGs and improve function in rodent models of SCI. To translate such treatments into the clinic, we need robust and sensitive methods for studying rodent models. In this study, we applied a newly developed suite of quantitative gait analysis tools: gait analysis instrumentation and technology optimized for rodents (GAITOR), which consists of an arena and open-source software (AGATHA: automated gait analysis through hues and areas). We showed that GAITOR can be used to detect subtle functional improvements (measured by hindlimb duty factor imbalance) in rats following ISP administration in a T10 hemisection injury model. We demonstrated that SCI-specific parameters (right paw placement accuracy and phase dispersion) can be easily added to GAITOR to track recovery. We confirmed the gait observations via retrograde tracer uptake. We concluded that GAITOR is a powerful tool for measuring recovery after moderate/mild SCI, and could be used to replace expensive/inflexible commercially-available gait analysis techniques.

Phytochemicals potently inhibit migration of metastatic breast cancer cells.

Cell migration is a major process that drives metastatic progression of cancers, the major cause of cancer death. Existing chemotherapeutic drugs have limited efficacy to prevent and/or treat metastasis, emphasizing the need for new treatments. We focus on triple negative breast cancer (TNBC), the subtype of breast cancer with worst prognosis and no standard chemotherapy protocols. Here we demonstrate that a group of natural compounds, known as phytochemicals, effectively block migration of metastatic TNBC cells. Using a novel cell micropatterning technology, we generate consistent migration niches in standard 96-well plates where each well contains a cell-excluded gap within a uniform monolayer of cells. Over time, cells migrate into and occupy the gap. Treating TNBC cells with non-toxic concentrations of phytochemicals significantly blocks motility of cells. Using a molecular analysis approach, we show that anti-migratory property of phytochemicals is partly due to their inhibitory effects on phosphorylation of ERK1/2. This study provides a framework for future studies to understand molecular targets of phytochemicals and evaluate their effectiveness in inhibiting metastasis in animal models of cancer.