Kevin Petrecca

Effects of Experimental Heart Failure on Atrial Cellular and Ionic Electrophysiology

BACKGROUNDnCongestive heart failure (CHF) is frequently associated with atrial fibrillation (AF), but little is known about the effects of CHF on atrial cellular electrophysiology.nnnMETHODS AND RESULTSnWe studied action potential (AP) properties and ionic currents in atrial myocytes from dogs with CHF induced by ventricular pacing at 220 to 240 bpm for 5 weeks. Atrial myocytes from CHF dogs were hypertrophied (mean+/-SEM capacitance, 89+/-2 pF versus 71+/-2 pF in control, n=160 cells per group, P<0.001). CHF significantly reduced the density of L-type Ca(2+) current (I(Ca)) by approximately 30%, of transient outward K(+) current (I(to)) by approximately 50%, and of slow delayed rectifier current (I(Ks)) by approximately 30% without altering their voltage dependencies or kinetics. The inward rectifier, ultrarapid and rapid delayed rectifier, and T-type Ca(2+) currents were not altered by CHF. CHF increased transient inward Na(+)/Ca(2+) exchanger (NCX) current by approximately 45%. The AP duration of atrial myocytes was not altered by CHF at slow rates but was increased at faster rates, paralleling in vivo refractory changes. CHF created a substrate for AF, prolonging mean AF duration from 8+/-4 to 535+/-82 seconds (P<0.01).nnnCONCLUSIONSnExperimental CHF selectively decreases atrial I(to), I(Ca), and I(Ks), increases NCX current, and leaves other currents unchanged. The cellular electrophysiological remodeling caused by CHF is quite distinct from that caused by atrial tachycardia, highlighting important differences in the cellular milieu characterizing different clinically relevant AF substrates.

N‐linked glycosylation sites determine HERG channel surface membrane expression

1 Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attributed to mutations in the human ether‐a‐go‐go‐related gene (HERG) that encodes a voltage‐gated cardiac K+ channel. While a recent report indicates that LQT in some patients is associated with a mutation of HERG at a consensus extracellular N‐linked glycosylation site (N629), earlier studies failed to identify a role for N‐linked glycosylation in the functional expression of voltage‐gated K+ channels. In this study we used pharmacological agents and site‐directed mutagenesis to assess the contribution of N‐linked glycosylation to the surface localization of HERG channels. 2 Tunicamycin, an inhibitor of N‐linked glycosylation, blocked normal surface membrane expression of a HERG‐green fluorescent protein (GFP) fusion protein (HERGGFP) transiently expressed in human embryonic kidney (HEK 293) cells imaged with confocal microscopy. 3 Immunoblot analysis revealed that N‐glycosidase F shifted the molecular mass of HERGGFP, stably expressed in HEK 293 cells, indicating the presence of N‐linked carbohydrate moieties. Mutations at each of the two putative extracellular N‐linked glycosylation sites (N598Q and N629Q) led to a perinuclear subcellular localization of HERGGFP stably expressed in HEK 293 cells, with no surface membrane expression. Furthermore, patch clamp analysis revealed that there was a virtual absence of HERG current in the N‐glycosylation mutants. 4 Taken together, these results strongly suggest that N‐linked glycosylation is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N‐linked glycosylation‐related mutations of HERG.

Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium

The Na+/H+ exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase alpha1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.The Na+/H+exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase α1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.

New prototype neuronavigation system based on preoperative imaging and intraoperative freehand ultrasound: system description and validation

PurposeThe aim of this report is to present IBIS (Interactive Brain Imaging System) NeuroNav, a new prototype neuronavigation system that has been developed in our research laboratory over the past decade that uses tracked intraoperative ultrasound to address surgical navigation issues related to brain shift. The unique feature of the system is its ability, when needed, to improve the initial patient-to-preoperative image alignment based on the intraoperative ultrasound data. Parts of IBIS Neuronav source code are now publicly available on-line.MethodsFour aspects of the system are characterized in this paper: the ultrasound probe calibration, the temporal calibration, the patient-to-image registration and the MRI-ultrasound registration. In order to characterize its real clinical precision and accuracy, the system was tested in a series of adult brain tumor cases.ResultsThree metrics were computed to evaluate the precision and accuracy of the ultrasound calibration. 1) Reproducibility: 1.77xa0mm and 1.65xa0mm for the bottom corners of the ultrasound image, 2) point reconstruction precision 0.62–0.90xa0mm: and 3) point reconstruction accuracy: 0.49–0.74xa0mm. The temporal calibration error was estimated to be 0.82xa0ms. The mean fiducial registration error (FRE) of the homologous-point-based patient-to-MRI registration for our clinical data is 4.9 ± 1.1xa0mm. After the skin landmark-based registration, the mean misalignment between the ultrasound and MR images in the tumor region is 6.1 ± 3.4xa0mm.ConclusionsThe components and functionality of a new prototype system are described and its precision and accuracy evaluated. It was found to have an accuracy similar to other comparable systems in the literature.

Sodium channel distribution within the rabbit atrioventricular node as analysed by confocal microscopy.

1 Paired 20 μm thick sections of fresh frozen tissue taken from the frontal plane of the rabbit atrioventricular (AV) nodal region were processed for histology and immunohistochemistry. Confocal microscopy was used to image the distribution of sodium channels using IgG (R12) developed against a highly conserved sequence in the interdomain 3–4 region of cloned sodium channels. 2 In ventricular and atrial cells, sodium channel immunofluorescence was localized to lateral membranes and T‐tubules. In the open AV node, levels of sodium channel immunofluorescence in the transitional cell zone and in the lower nodal cell tract were comparable to that found in the atrial and ventricular myocardium. 3 In the enclosed AV node a gradation of sodium channel immunofluorescence is present such that peripherally located circumferential transitional cells display high levels of immunofluorescence, comparable to that of atrial and ventricular myocardium, while centrally located midnodal cells display decreased levels of or no immunofluorescence. 4 In order to correlate the distribution of sodium channels with the distribution of gap junctions, we used IgG directed against the carboxyl terminus of connexin43 (CT‐360). Ventricular cell immunofluorescence was localized primarily to the intercalated disk region, while in the AV node, the pattern of distribution was found to be similar to that of sodium channels. 5 The reduced levels of and/or absence of immunofluorescence in the midnodal cell region indicates a paucity of sodium channel and connexin43 protein expression in this region of the AV node that would favour slow impulse conduction.

Comparing two approaches to rigid registration of three-dimensional ultrasound and magnetic resonance images for neurosurgery

PurposeWe present a new technique for registering magnetic resonance (MR) and ultrasound images in the context of neurosurgery. It involves generating a pseudo-ultrasound (pseudo-US) from a segmented MR image and uses cross-correlation as the cost function to register the pseudo-US to the real ultrasound data. The algorithm’s performance is compared with that of a state-of-the-art technique that uses a median-filtered MR image to register to a Gaussian-blurred ultrasound using a normalized mutual information (NMI) objective function.MethodsThe two methods were tested on data from 15 patients with brain tumor, including low-and high-grade gliomas, in both first operations and reoperations. Two metrics were used to evaluate registration accuracy: (1) the mean distance between corresponding points, identified on both MR and ultrasound images by two experts, and (2) ratings based on visual comparison by one neurosurgeon.ResultsThe mean residual distance of the pseudo-US technique, 2.97xa0mm, is significantly more accurate (pxa0=xa0.0011) than that of the NMI approach, 4.86xa0mm. The visual assessment shows that only 4 of the 15 cases had a satisfactory initial alignment based on homologous skin-point registration. There is a significant correlation between the quantitative distance measures and the qualitative ratings (rho =xa00.785).ConclusionThe results show that the pseudo-US rigid registration technique robustly improves the MRI–ultrasound alignment when compared with the initial alignment, even when applied to highly distorted brains and a large range of tumor sizes and appearances.

Brain shift in neuronavigation of brain tumors: A review

Purpose: Neuronavigation based on preoperative imaging data is a ubiquitous tool for image guidance in neurosurgery. However, it is rendered unreliable when brain shift invalidates the patient‐to‐image registration. Many investigators have tried to explain, quantify, and compensate for this phenomenon to allow extended use of neuronavigation systems for the duration of surgery. The purpose of this paper is to present an overview of the work that has been done investigating brain shift. Methods: A review of the literature dealing with the explanation, quantification and compensation of brain shift is presented. The review is based on a systematic search using relevant keywords and phrases in PubMed. The review is organized based on a developed taxonomy that classifies brain shift as occurring due to physical, surgical or biological factors. Results: This paper gives an overview of the work investigating, quantifying, and compensating for brain shift in neuronavigation while describing the successes, setbacks, and additional needs in the field. An analysis of the literature demonstrates a high variability in the methods used to quantify brain shift as well as a wide range in the measured magnitude of the brain shift, depending on the specifics of the intervention. The analysis indicates the need for additional research to be done in quantifying independent effects of brain shift in order for some of the state of the art compensation methods to become useful. Conclusion: This review allows for a thorough understanding of the work investigating brain shift and introduces the needs for future avenues of investigation of the phenomenon. HighlightsA comprehensive review of research on the phenomenon of brain shift.A new taxonomy separating brain shift into physical, biological and surgical factors.Contrast between brain shift corrections through intraoperative imaging methods.Recommendations for future focus of brain shift research. Graphical abstract Figure. Image, graphical abstract

Spatial distribution of nerve processes and β-adrenoreceptors in the rat atrioventricular node

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene‐related peptide (CGRP) and β1 and β2‐adrenoreceptors. Serial 12 μm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE‐reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE‐reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH‐immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. β1‐adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. β2‐adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of β2‐ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype‐specific pharmacological agents may have distinct effects upon AV nodal conduction.

Rigid Registration of 3D Ultrasound and MRI: Comparing Two Approaches on Nine Tumor Cases

We present a new technique for registering ultrasound and magnetic resonance (MR) images in the context of neurosurgery. It involves generating a pseudo-ultrasound (pseudo-US) from a segmented MR image and uses cross-correlation as the cost function to register with ultrasound. The algorithm’s performance is compared to a state-of-the-art technique that uses a median filtered MR images to register with a Gaussian-blurred ultrasound using normalized mutual information (NMI). The two methods are tested on nine tumor cases, including both high- and low-grade gliomas. The pseudo-US method yielded significantly better alignment average than that obtained by NMI (p = 0.0009). If one case where NMI failed is excluded, the mean distance obtained by the pseudo-US approach (2.6 mm) is slightly lower than the one obtained by NMI (2.8mm), but not significantly so (p = 0.16). We conclude that the pseudo-US method is more robust for these cases.

A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer

Therapies targeting epidermal growth factor receptor (EGFR) have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention.