Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Using NSQIP Data to Reduce Institutional Postoperative Pneumonia Rates in Non-ICU Patients: A Plan-Do-Study-Act Approach.

BACKGROUND: The American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) is a program designed to measure and improve surgical care quality. In 2015, the study institution formed a multidisciplinary team to address the poor adult postoperative pneumonia performance (worst decile). STUDY DESIGN: The study institution is a 450+ bed tertiary care center that performs 12,000+ surgical procedures annually. From January 2016 to December 2019, the institution abstracted surgical cases and assigned postoperative pneumonia as a complication per the NSQIP operations manual. Using a plan-do-study-act approach, a multidisciplinary postoperative pneumonia prevention team implemented initiatives regarding incentive spirometry education, anesthetic optimization, early mobility, and oral care. The team measured the initiatives' success by analyzing semiannual reports (SAR) provided by the ACS NSQIP and regional adjusted percentile rankings provided by the Georgia Surgical Quality Collaborative (GSQC). RESULTS: The 2015 SAR postoperative pneumonia rate was 4.20% (odds ratio [OR] 3.86, confidence interval [CI] 2.92-5.11). After project initiation, the postoperative pneumonia rates decreased for all NSQIP cases, from 2.51% (OR 2.67, CI 1.89-3.77) in 2016 to 2.08% (OR 2.61, CI 1.82-3.74) in 2017, to 0.85% (OR 1.10, CI 0.69-1.75) in 2018, and then increased slightly to 1.14% (OR 1.27, CI 0.84-1.92) in 2019. The institution's adjusted percentile regional rank of participating regional ACS NSQIP hospitals' postoperative pneumonia rate improved from 14/14 (July 2015-June 2016) to 6/14 (July 2018-June 2019). CONCLUSIONS: The multidisciplinary postoperative pneumonia prevention team successfully decreased the postoperative pneumonia rate, therefore improving surgical patients' outcomes. Furthermore, this quality improvement project also saved valuable revenue for the hospital.

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.].

Ventricular Endocardial Tissue Geometry Affects Stimulus Threshold and Effective Refractory Period.

BACKGROUND: Understanding the biophysical processes by which electrical stimuli applied to cardiac tissue may result in local activation is important in both the experimental and clinical electrophysiology laboratory environments, as well as for gaining a more in-depth knowledge of the mechanisms of focal-trigger-induced arrhythmias. Previous computational models have predicted that local myocardial tissue architecture alone may significantly modulate tissue excitability, affecting both the local stimulus current required to excite the tissue and the local effective refractory period (ERP). In this work, we present experimental validation of this structural modulation of local tissue excitability on the endocardial tissue surface, use computational models to provide mechanistic understanding of this phenomena in relation to localized changes in electrotonic loading, and demonstrate its implications for the capture of afterdepolarizations. METHODS AND RESULTS: Experiments on rabbit ventricular wedge preparations showed that endocardial ridges (surfaces of negative mean curvature) had a stimulus capture threshold that was 0.21 ± 0.03 V less than endocardial grooves (surfaces of positive mean curvature) for pairwise comparison (24% reduction, corresponding to 56.2 ± 6.4% of the energy). When stimulated at the minimal stimulus strength for capture, ridge locations showed a shorter ERP than grooves (n = 6, mean pairwise difference 7.4 ± 4.2 ms). When each site was stimulated with identical-strength stimuli, the difference in ERP was further increased (mean pairwise difference 15.8 ± 5.3 ms). Computational bidomain models of highly idealized cylindrical endocardial structures qualitatively agreed with these findings, showing that such changes in excitability are driven by structural modulation in electrotonic loading, quantifying this relationship as a function of surface curvature. Simulations further showed that capture of delayed afterdepolarizations was more likely in trabecular ridges than grooves, driven by this difference in loading. CONCLUSIONS: We have demonstrated experimentally and explained mechanistically in computer simulations that the ability to capture tissue on the endocardial surface depends upon the local tissue architecture. These findings have important implications for deepening our understanding of excitability differences related to anatomical structure during stimulus application that may have important applications in the translation of novel experimental optogenetics pacing strategies. The uncovered preferential vulnerability to capture of afterdepolarizations of endocardial ridges, compared to grooves, provides important insight for understanding the mechanisms of focal-trigger-induced arrhythmias.

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.

Normal interventricular differences in tissue architecture underlie right ventricular susceptibility to conduction abnormalities in a mouse model of Brugada syndrome.

Aims: Loss-of-function of the cardiac sodium channel NaV1.5 is a common feature of Brugada syndrome. Arrhythmias arise preferentially from the right ventricle (RV) despite equivalent NaV1.5 downregulation in the left ventricle (LV). The reasons for increased RV sensitivity to NaV1.5 loss-of-function mutations remain unclear. Because ventricular electrical activation occurs predominantly in the transmural axis, we compare RV and LV transmural electrophysiology to determine the underlying cause of the asymmetrical conduction abnormalities in Scn5a haploinsufficient mice (Scn5a+/-). Methods and results: Optical mapping and two-photon microscopy in isolated-perfused mouse hearts demonstrated equivalent depression of transmural conduction velocity (CV) in the LV and RV of Scn5a+/- vs. wild-type littermates. Only RV transmural conduction was further impaired when challenged with increased pacing frequencies. Epicardial dispersion of activation and beat-to-beat variation in activation time were increased only in the RV of Scn5a+/- hearts. Analysis of confocal and histological images revealed larger intramural clefts between cardiomyocyte layers in the RV vs. LV, independent of genotype. Acute sodium current inhibition in wild type hearts using tetrodotoxin reproduced beat-to-beat activation variability and frequency-dependent CV slowing in the RV only, with the LV unaffected. The influence of clefts on conduction was examined using a two-dimensional monodomain computational model. When peak sodium channel conductance was reduced to 50% of normal the presence of clefts between cardiomyocyte layers reproduced the activation variability and conduction phenotype observed experimentally. Conclusions: Normal structural heterogeneities present in the RV are responsible for increased vulnerability to conduction slowing in the presence of reduced sodium channel function. Heterogeneous conduction slowing seen in the RV will predispose to functional block and the initiation of re-entrant ventricular arrhythmias.

Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells.

Myocardial injury results in a loss of contractile tissue mass that, in the absence of efficient regeneration, is essentially irreversible. Transplantation of human pluripotent stem cell-derived cardiomyocytes has beneficial but variable effects. We created human engineered heart tissue (hEHT) strips from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived endothelial cells. The hEHTs were transplanted onto large defects (22% of the left ventricular wall, 35% decline in left ventricular function) of guinea pig hearts 7 days after cryoinjury, and the results were compared with those obtained with human endothelial cell patches (hEETs) or cell-free patches. Twenty-eight days after transplantation, the hearts repaired with hEHT strips exhibited, within the scar, human heart muscle grafts, which had remuscularized 12% of the infarct area. These grafts showed cardiomyocyte proliferation, vascularization, and evidence for electrical coupling to the intact heart tissue in a subset of engrafted hearts. hEHT strips improved left ventricular function by 31% compared to that before implantation, whereas the hEET or cell-free patches had no effect. Together, our study demonstrates that three-dimensional human heart muscle constructs can repair the injured heart.

2-Photon excitation fluorescence microscopy enables deeper high-resolution imaging of voltage and Ca(2+) in intact mice, rat, and rabbit hearts.

We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (Vm ) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca(2+) recordings. Action potentials (APs) were accurately characterized by 2PLSM vs. microelectrodes, albeit fast events (<1 ms) were sub-optimally acquired by 2PLSM due to limited sampling frequencies (2.6 kHz). The slower Ca(2+) transient (CaT) time course (>1ms) could be accurately described by 2PLSM. In conclusion, Vm - and Ca(2+) -sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca(2+) signals to ∼400 µm.

Subepicardial action potential characteristics are a function of depth and activation sequence in isolated rabbit hearts.

BACKGROUND: Electric excitability in the ventricular wall is influenced by cellular electrophysiology and passive electric properties of the myocardium. Action potential (AP) rise time, an indicator of myocardial excitability, is influenced by conduction pattern and distance from the epicardial surface. This study examined AP rise times and conduction velocity as the depolarizing wavefront approaches the epicardial surface. METHODS AND RESULTS: Two-photon excitation of di-4-aminonaphthenyl-pyridinum-propylsulfonate was used to measure electric activity at discrete epicardial layers of isolated Langendorff-perfused rabbit hearts to a depth of 500 µm. Endo-to-epicardial wavefronts were studied during right atrial or ventricular endocardial pacing. Similar measurements were made with epi-to-endocardial, transverse, and longitudinal pacing protocols. Results were compared with data from a bidomain model of 3-dimensional (3D) electric propagation within ventricular myocardium. During right atrial and endocardial pacing, AP rise time (10%-90% of upstroke) decreased by ≈50% between 500 and 50 µm from the epicardial surface, whereas conduction velocity increased and AP duration was only slightly shorter (≈4%). These differences were not observed with other conduction patterns. The depth-dependent changes in rise time were larger at higher pacing rates. Modeling data qualitatively reproduced the behavior seen experimentally and demonstrated a parallel reduction in peak I(Na) and electrotonic load as the wavefront approaches the epicardial surface. CONCLUSIONS: Decreased electrotonic load at the epicardial surface results in more rapid AP upstrokes and higher conduction velocities compared with the bulk myocardium. Combined effects of tissue depth and pacing rate on AP rise time reduce conduction safety and myocardial excitability within the ventricular wall.

Isolated rabbit working heart function during progressive inhibition of myocardial SERCA activity.

RATIONALE: The extent to which sarcoplasmic reticulum Ca(2+)ATPase (SERCA) activity alone determines left ventricular (LV) pump function is unknown. OBJECTIVE: To correlate SERCA activity with hemodynamic function of rabbit LV during thapsigargin perfusion. METHODS AND RESULTS: Isolated rabbit hearts were perfused in working heart configuration, and LV pump function was assessed using a pressure-volume catheter. Rapid and complete (>95%) inhibition of SERCA was associated with a moderate decrease in cardiac function (to 70%-85% of control). Further decrease in cardiac function to 50%-75% of control occurred over the next ≈ 30 minutes despite no detectable further inhibition of SERCA activity. Analysis of the 20 seconds prior to pump failure revealed a rapid decrease in end diastolic volume. Intermediate levels of SERCA function (≈ 50% of control) had only minor hemodynamic effects. Parallel experiments in field-stimulated isolated ventricular cardiomyocytes monitored intracellular Ca(2+) and cell shortening. On perfusion with thapsigargin, Ca(2+) transient amplitude and cell shortening fell to ≈ 70% of control followed by increased diastolic Ca(2+) concentration and diastolic cell shortening to achieve a new steady state. CONCLUSIONS: The relationship between SERCA activity and LV function in the rabbit is highly nonlinear. In the short term, only moderate effects on LV pump function were observed despite almost complete (>95%) reduction in SERCA activity. The terminal decline of function was associated with sudden sustained increase in diastolic tone comparable to the sustained contraction observed in isolated cardiomyocytes. Secondary increases of intracellular Ca(2+) and Na(+) following complete SERCA inhibition eventually limit contractile function and precipitate LV pump failure.