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 use 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.NEW & NOTEWORTHY Rodent models are widely used in arrhythmia research. However, most studies do not standardize clinically relevant in vivo and ex vivo techniques to support their conclusions. Here, we present a comprehensive electrophysiological protocol in an arrhythmogenic rat model, connecting in vivo and ex vivo programmed electrical stimulation with optical mapping. By establishing this protocol, we aim to facilitate the adoption of a standardized model for investigating arrhythmias, enhancing research rigor and comparability in this field.

Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction.

AIMS: Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. METHODS AND RESULTS: Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these "exercise-miRNAs" individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the "exercise-miRNAs", miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. CONCLUSION: Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.

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

BACKGROUND & 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.

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

[This corrects the article DOI: 10.3389/fphys.2018.01454.].

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.

Aerobic Interval Training Prevents Age-Dependent Vulnerability to Atrial Fibrillation in Rodents.

Aims: Increasing age is the most important risk factor for atrial fibrillation (AF). Very high doses of exercise training might increase AF risk, while moderate levels seem to be protective. This study aimed to examine the effects of age on vulnerability to AF and whether long-term aerobic interval training (AIT) could modify these effects. Methods: Nine months old, male Sprague-Dawley rats were randomized to AIT for 16 weeks (old-ex) or to a sedentary control group (old-sed), and compared to young sedentary males (young-sed). After the intervention, animals underwent echocardiography, testing of exercise capacity (VO2max), and electrophysiology with AF induction before ex vivo electrophysiology. Fibrosis quantification, immunohistochemistry and western blotting of atrial tissue were performed. Results: Sustained AF was induced in vivo in 4 of 11 old-sed animals, but none of the old-ex or young-sed rats (p = 0.006). VO2max was lower in old-sed, while old-ex had comparable results to young-sed. Fibrosis was increased in old-sed (p = 0.006), with similar results in old-ex. There was a significantly slower atrial conduction in old-sed (p = 0.038), with an increase in old-ex (p = 0.027). Action potential duration was unaltered in old-sed, but prolonged in old-ex (p = 0.036). There were no differences in amount of atrial connexin 43 between groups, but a lateralization in atrial cardiomyocytes of old-sed, with similar findings in old-ex. Conclusion: AF vulnerability was higher in old-sed animals, associated with increased atrial fibrosis, lateralization of connexin-43, and reduced atrial conduction velocity. AIT reduced the age-associated susceptibility to AF, possibly through increased conduction velocity and prolongation of action potentials.

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.

Acute exhaustive aerobic exercise training impair cardiomyocyte function and calcium handling in Sprague-Dawley rats.

INTRODUCTION: Recent data from long-distance endurance participants suggest that cardiac function is impaired after completion. Existing data further indicate that right ventricular function is more affected than left ventricular function. The cellular mechanisms underpinning cardiac deterioration are limited and therefore the aim of this study was to examine cardiomyocyte and molecular responses of the right and left ventricle to an acute bout of exhaustive endurance exercise. MATERIALS AND METHODS: Male Sprague-Dawley rats were assigned to sedentary controls or acute exhaustive endurance exercise consisting of a 120 minutes long forced treadmill run. The contractile function and Ca2+ handling properties in isolated cardiomyocytes, protein expression levels of sarcoplasmic reticulum Ca2+-ATPase and phospholamban including two of its phosphorylated states (serine 16 and threonine 17), and the mitochondrial respiration in permeabilized cardiac muscle fibers were analyzed. RESULTS: The exercise group showed a significant reduction in cardiomyocyte fractional shortening (right ventricle 1 Hz and 3 Hz p<0.001; left ventricle 1 Hz p<0.05), intracellular Ca2+ amplitude (right ventricle 1 and 3 Hz p<0.001; left ventricle 1 Hz p<0.01 and 3 Hz p<0.05) and rate of diastolic Ca2+ decay (right ventricle 1 Hz p<0.001 and 3 Hz p<0.01; left ventricle 1 and 3 Hz p<0.01). Cardiomyocyte relaxation during diastole was only significantly prolonged at 3 Hz in the right ventricle (p<0.05) compared to sedentary controls. We found an increase in phosphorylation of phospholamban at serine 16 and threonine 17 in the left (p<0.05), but not the right, ventricle from exhaustively exercised animals. The protein expression levels of sarcoplasmic reticulum Ca2+-ATPase and phospholamban was not changed. Furthermore, we found a reduction in maximal oxidative phosphorylation and electron transport system capacities of mitochondrial respiration in the right (p<0.01 and p<0.05, respectively), but not the left ventricle from rats subjected to acute exhaustive treadmill exercise. CONCLUSION: Acute exhaustive treadmill exercise is associated with impairment of cardiomyocyte Ca2+ handling and mitochondrial respiration that causes depression in both contraction and diastolic relaxation of cardiomyocytes.

X-ray repair cross complementing protein 1 in base excision repair.

X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.

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 166-436 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 POLß 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.

XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage.

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil.