Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart

Background and aims: Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. Methods: Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. Results: FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. Conclusion: Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties

Characterization of electrical activity in post-myocardial infarction scar tissue in rat hearts using multiphoton microscopy.

Background: The origin of electrical behavior in post-myocardial infarction scar tissue is still under debate. This study aims to examine the extent and nature of the residual electrical activity within a stabilized ventricular infarct scar. Methods and Results: An apical infarct was induced in the left ventricle of Wistar rats by coronary artery occlusion. Five weeks post-procedure, hearts were Langendorff-perfused, and optically mapped using di-4-ANEPPS. Widefield imaging of optical action potentials (APs) on the left ventricular epicardial surface revealed uniform areas of electrical activity in both normal zone (NZ) and infarct border zone (BZ), but only limited areas of low-amplitude signals in the infarct zone (IZ). 2-photon (2P) excitation of di-4-ANEPPS and Fura-2/AM at discrete layers in the NZ revealed APs and Ca2+ transients (CaTs) to 500-600 μm below the epicardial surface. 2P imaging in the BZ revealed superficial connective tissue structures lacking APs or CaTs. At depths greater than approximately 300 μm, myocardial structures were evident that supported normal APs and CaTs. In the IZ, although 2P imaging did not reveal clear myocardial structures, low-amplitude AP signals were recorded at discrete layers. No discernible Ca2+ signals could be detected in the IZ. AP rise times in BZ were slower than NZ (3.50 ± 0.50 ms vs. 2.23 ± 0.28 ms) and further slowed in IZ (9.13 ± 0.56 ms). Widefield measurements of activation delay between NZ and BZ showed negligible difference (3.37 ± 1.55 ms), while delay values in IZ showed large variation (11.88 ± 9.43 ms). Conclusion: These AP measurements indicate that BZ consists of an electrically inert scar above relatively normal myocardium. Discrete areas/layers of IZ displayed entrained APs with altered electrophysiology, but the structure of this tissue remains to be elucidated

Corrigendum: Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy

No abstract available

A comprehensive protocol combining in vivo and ex vivo electrophysiological experiments in an arrhythmogenic animal model

Ventricular arrhythmias contribute significantly to cardiovascular mortality, with coronary artery disease as the predominant underlying cause. Understanding the mechanisms of arrhythmogenesis is essential to identify proarrhythmic factors and develop novel approaches for antiarrhythmic prophylaxis and treatment. Animal models are vital in basic research on cardiac arrhythmias, encompassing molecular, cellular, ex vivo whole heart and in vivo models. Most studies employ either in vivo protocols lacking important information on clinical relevance, or exclusively ex vivo protocols, thereby missing the opportunity to explore underlying mechanisms. Consequently, interpretation may be difficult due to dissimilarities in animal models, interventions, and individual properties across animals. Moreover, proarrhythmic effects observed in vivo are often not replicated in corresponding ex vivo preparations during mechanistic studies. We have established a protocol to perform both an in vivo and ex vivo electrophysiological characterization in an arrhythmogenic rat model with heart failure following myocardial infarction. The same animal is followed throughout the experiment. In vivo methods involve intracardiac programmed electrical stimulation and external defibrillation to terminate sustained ventricular arrhythmia. Ex vivo methods conducted on the Langendorff-perfused heart include an electrophysiological study with optical mapping of regional action potentials, conduction velocities, and dispersion of electrophysiological properties. By exploring the retention of the in vivo proarrhythmic phenotype ex vivo, we aim to examine whether the subsequent ex vivo detailed measurements are relevant to in vivo pathological behavior. This protocol can enhance greater understanding of cardiac arrhythmias by providing a standardized, yet adaptable model for evaluating arrhythmogenicity or antiarrhythmic interventions in cardiac diseases

A Historical Perspective of Influenza A(H1N2) Virus

The emergence and transition to pandemic status of the influenza A(H1N1)A(H1N1)pdm09) virus in 2009 illustrated the potential for previously circulating human viruses to re-emerge in humans and cause a pandemic after decades of circulating among animals. Within a short time of the initial emergence of A(H1N1)pdm09 virus, novel reassortants were isolated from swine. In late 2011, a variant (v) H3N2 subtype was isolated from humans, and by 2012, the number of persons infected began to increase with limited person-to-person transmission. During 2012 in the United States, an A(H1N2)v virus was transmitted to humans from swine. During the same year, Australia recorded its first H1N2 subtype infection among swine. The A(H3N2)v and A(H1N2)v viruses contained the matrix protein from the A(H1N1)pdm09 virus, raising the possibility of increased transmissibility among humans and underscoring the potential for influenza pandemics of novel swine-origin viruses. We report on the differing histories of A(H1N2) viruses among humans and animals

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

P38 MAPK signaling and phosphorylations in the BRCT1 domain regulate XRCC1 recruitment to sites of DNA damage

XRCC1 is a scaffold protein involved in base excision repair and single strand break repair. It is a phosphoprotein that contains more than 45 phosphorylation sites, however only a few of these have been characterized and connected to specific kinases and functions. Mitogen activated protein kinases (MAPK) are mediators of cellular stress responses, and here we demonstrate that p38 MAPK signaling is involved in phosphorylation of XRCC1 and regulation of recruitment to oxidative stress. Inhibition of p38 MAPK caused a marked pI shift of XRCC1 towards a less phosphorylated state. Inhibition of p38 also increased the immediate accumulation of XRCC1 at site of DNA damage in a poly(ADP)-ribose (PAR) dependent manner. These results suggest a link between PARylation, p38 signaling and XRCC1 recruitment to DNA damage. Additionally, we characterized two phosphorylation sites, T358 and T367, located within, or close to, the phosphate-binding pocket of XRCC1, which is important for interaction with PAR. Mutation of these sites impairs recruitment of XRCC1 to DNA damage and binding to PARP1/PAR. Collectively, our data suggest that phosphorylation of T358 and T367 and p38 signaling are important for proper regulation of XRCC1 recruitment to DNA damage and thereby avoidance of potential toxic and mutagenic BER-intermediates

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1

XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166 to 436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLβ or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein

Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy

Background: The origin of electrical behavior in post-myocardial infarction scar tissue is still under debate. This study aims to examine the extent and nature of the residual electrical activity within a stabilized ventricular infarct scar. Methods and Results: An apical infarct was induced in the left ventricle of Wistar rats by coronary artery occlusion. Five weeks post-procedure, hearts were Langendorff-perfused, and optically mapped using di-4-ANEPPS. Widefield imaging of optical action potentials (APs) on the left ventricular epicardial surface revealed uniform areas of electrical activity in both normal zone (NZ) and infarct border zone (BZ), but only limited areas of low-amplitude signals in the infarct zone (IZ). 2-photon (2P) excitation of di-4-ANEPPS and Fura-2/AM at discrete layers in the NZ revealed APs and Ca2+ transients (CaTs) to 500–600 μm below the epicardial surface. 2P imaging in the BZ revealed superficial connective tissue structures lacking APs or CaTs. At depths greater than approximately 300 μm, myocardial structures were evident that supported normal APs and CaTs. In the IZ, although 2P imaging did not reveal clear myocardial structures, low-amplitude AP signals were recorded at discrete layers. No discernible Ca2+ signals could be detected in the IZ. AP rise times in BZ were slower than NZ (3.50 ± 0.50 ms vs. 2.23 ± 0.28 ms) and further slowed in IZ (9.13 ± 0.56 ms). Widefield measurements of activation delay between NZ and BZ showed negligible difference (3.37 ± 1.55 ms), while delay values in IZ showed large variation (11.88 ± 9.43 ms). Conclusion: These AP measurements indicate that BZ consists of an electrically inert scar above relatively normal myocardium. Discrete areas/layers of IZ displayed entrained APs with altered electrophysiology, but the structure of this tissue remains to be elucidated