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The right-ventricular (RV) outflow tract (RVOT) and the transition to the RV free wall are recognized sources of arrhythmia in human hearts. However, we do not fully understand myocardial tissue structures in this region. Human heart tissue was processed for optical clarity, labelled with wheat-germ agglutin (WGA) and anti-Cx43, and imaged on a custom-built line scanning confocal microscope. The 3D images were analyzed for myocyte gross structures and cell morphology. There were regions of high organization as well as rapid changes to more heterogeneous regions. Preliminary cell segmentations were used to estimate cell morphology. Observed RVOT/RV structure is consistent with known arrhythmic substrates.Clinical Relevance- New views of human tissue structure enable clearer clinical understanding of arrhythmogenic activation pathways and targets for invasive treatment such as RF ablation.

Heart rate variability (HRV) is an important clinical parameter that depicts the autonomic balance. Diminished HRV has been associated with diseased hearts and incorporating stochasticity in pacing has been investigated as a potential mechanism for restoring the altered autonomic balance and preventing cardiac arrhythmias. We studied the change in HRV with the development of chronic myocardial infarction (MI) in adult sheep (n=16). Next, we investigated the utility of stochastic pacing in modulating HRV in-vivo in both sham and MI hearts. The propensity of the heart to the development of cardiac alternans, a known precursor to tachyarrhythmias, was studied under three different pacing techniques, namely periodic pacing, stochastic pacing and constant diastolic interval (DI) pacing in one sham and one MI sheep. Autonomic balance was observed to be altered after 6 weeks of chronic MI. Increased heart rate, QTc interval, standard deviation of the R-R intervals and LF/HF ratio was observed in MI hearts. Stochastic pacing was found to be proarrhythmic and increased T-wave alternans burden was observed with increase in stochasticity. Maintaining a constant DI on every beat demonstrated reduced alternans levels compared to both periodic and stochastic pacing.Clinical Relevance-Our results demonstrate that precise control of the diastolic interval may be more beneficial in inhibiting arrhythmias than stochastic pacing.

Cardiac alternans has been associated with an increased propensity to lethal tachyarrhythmias such as ventricular tachycardia and fibrillation (VT/VF). Myocardial infarction (MI), resulting from restricted oxygen supply to the heart, is a known substrate for VT/VF. Here, we investigate the utility of cardiac alternans as a predictor of tachyarrhythmias in a chronic MI ovine model. In-vivo electrophysiological studies were performed to assess the change in microvolt T-wave alternans (TWA) with induction of acute ischemia following coronary artery occlusion. 24-hour telemetry was performed in an ambulatory animal for 6 weeks to monitor the progression of TWA with chronic MI. At 6 weeks, ex-vivo optical mapping experiments were performed to assess the spatiotemporal evolution of alternans in sham (n=5) and chronic MI hearts (n=8). Our results demonstrate that chronic MI leads to significant electrophysiological changes in the cardiac substrate. Significant increase in TWA is observed post occlusion and a steady rise in alternans is seen with progression of chronic MI. Compared to sham, chronic MI hearts show significant presence of localized action potential amplitude alternans, which spatially evolve with an increase in pacing frequency. Clinical Relevance - Our results demonstrate that localized alternans underlie arrhythmogenesis in chronic MI hearts and microvolt TWA can serve as a biomarker of disease progression during chronic MI.

Pathological cardiac fibrosis is important in predisposing the heart to arrhythmia and mechanical failure. The regional distribution of fibrosis is often described qualitatively and quantitatively in histological studies of animal hearts after staining collagen with specific colored stains. Currently this description is often piecemeal, as it lacks rigorous spatial registration, matching and methodological standardization between animals and between study groups. We propose a strategy for the quantification of regional fibrosis using the American Heart Association (AHA) cardiac segmentation model. We quantify fibrosis after whole heart 3D histological reconstruction in one normal rat heart and in one rat heart in right heart failure induced by monocrotaline. We then assess the minimum spaced histological sampling which allows for accurate assessment of regional fibrosis. We show that using every section of a set of 5 μm serial sections quantifies regional right ventricular fibrosis, with highly significant (p <; 0.001) differences between heart failure and control hearts. We show that the absolute error of collagen quantification is low when sections are taken spaced by up to 100 μm (error 5.7±5.8%). Likewise, absolute error associated with sectioning starting position is low for sections spaced up to 100 μm (error 13.3±17.2%). Above 100 μm section spacing quantification error is large (tending to 50%) and error associated with sectioning starting position is large (tending to 60%).

DT-MRI has been widely used to quantify myocardial fiber and laminar orientations. These structural orientations influence both the spread of excitation and the reorganization of the myocardium during contraction and are altered in disease states. Studies have sought to validate DT-MRI but questions remain about the accuracy of the method and its sensitivity to the time post-fixation and imaging parameters, including b-value, number of diffusion directions and image voxel size. The advent of high-spatial resolution ex vivo MRI and structure tensor (ST) analysis provides a means of direct validation of DT-MRI and assessment of sensitivity to the b-value, the number of diffusion directions and the image voxel size. We find that, with the fixation method we used, structure does not change with time (up to 72 hours). We show that DT-MRI and ST/HR-MRI are markedly similar measures of fiber orientation but DT-MRI and ST are much less similar measures of laminar orientation. DT-MRI performance is not sensitive to the number of directions, with similar structural orientations measured with 6 or 12 directions. Likewise, DT-MRI performance is generally insensitive to b-value, but laminar measurement is moderately more accurate at b = 500 than for higher b-values.

Cardiac arrhythmias are often characterized by non-repetitive complex activation sequences. The properties of electrical activity in cardiac tissue, such as activation time (AT), can be accurately determined using optical imaging of electrical signals using voltage-sensitive dyes. The electrical AT of optical action potentials is known to accurately correlate with the time of the maximal derivative (dF/dt(max)) of the upstroke of ensemble averaged optical signals. The sensitivity of dF/dt(max) to noise is therefore problematic for mapping AT from nonrepetitive activity. Here we experimentally evaluated a novel method to determine activation times (AT) from non-averaged epicardial optical signals. The method depends upon the time delays of activation between adjacent pixels developing a global AT map, as opposed to measuring local AT from each pixel independently based on dF/dt(max). ATs from dF/dt(max) and global AT maps of non-ensemble averaged signals were correlated with averaged signals. Global ATs improved linear correlation coefficients and accuracy of AT patterns. Furthermore, global AT maps were significantly more robust at reproducing AT maps between consecutive stable beats. The proposed method of global AT mapping will potentially enable accurate mapping of non-repetitive propagation during arrhythmias.

The ventricular myocardium has a structure of branching laminae through which course regularly orientated myofibers, an architecture important in excitation and contraction. Quantifying this architecture is vital for understanding normal and disease states in the heart and for assessing their impact on electrical function. These data are also highly important in the construction of scientifically and clinically useful computer models of cardiac electrical behavior. Detailed structural information has previously been obtained from serial imaging. In this work we assess the potential for high-resolution (HR) MRI as a means to furnish useful myoarchitecture and compare and contrast this approach with the growing use of DT. Using rat hearts, we conclude that both approaches have strengths and weaknesses, however, HR-MRI may provide a consistently more robust picture of the myoarchitecture in small hearts.

Transmural myocardial activation is influenced by myocardial structure, including structural differences between the compacta (Cta) and the trabeculata (Tta), although this has not been fully explained. Hearts from rats were Langendorff perfused, stained with DI-4-ANEPPS, the apex was cut off and fluorescence acquired from the exposed short-axis surface. The hearts were stimulated at 160 ms cycle length at the anterior, lateral, posterior left ventricle (LV) and septal sub-epicardial sites. Conduction velocity perpendicular to the wave front orientation was measured in each pixel using a gradient-based approach. After optical mapping the cut surface was imaged using a light microscope and the extent of the Cta and Tta mapped and validated against 50 u, m isotropic MRI images. We used a 3D rat ventricle computational model, with architecture obtained from 200 u, m isotropic diffusion tensor MRI and kinetics from the modified Pandit model to determine the relative roles of fibers and sheets on propagation. We show in the experimental study that circumferential propagation around the LV cavity is fast in the Cta: 63.2±19.5 and is slower in the Tta: 32.7±11.0(∗) (mean ± s.d cms-1, ∗ p < 0.01 by two sample t test). In the simulation study the pattern and velocity are not replicated in an isotropic model (I), are partially replicated in a simulation study including fiber anisotropy (A) and is more fully replicated in orthotropic (O) ventricles (fiber and sheet anisotropy), where the circumferential propagation velocity is, I: Cta: 54.2±3.9; Tta:54.3±3.9; A: Cta:43.6±3.2; Tta: 40.6±6.6; O: Cta: 63.2±19.5; Tta: 32.7±11.9(∗). We show that sheet orientation is important in understanding activation differences between Cta and Tta.

Current techniques to map intramural activation patterns in ex vivo cardiac tissue have limited spatial resolution. Here, we report on the experimental validation of a novel optical technique that was recently proposed to resolve the size and depth of intramural wave fronts using alternating transillumination (AT). AT was achieved by simultaneously mapping the epi- and endocardial surfaces with two synchronized CCD cameras and rapidly alternating LED illumination between both surfaces. Optical phantoms were made based on tissue optical properties measured using a hybrid optical spectrometer. Spherical fluorescent sources (Scarlet microspheres, Invitrogen, UK) of varying sizes were embedded at known depths in the phantoms. Coronary-perfused procine left ventricular slab preparations were stained with DI-4-ANBDQBS (n = 3) and paced at known intramural depths. In phantoms we were able to reliably estimate the depth of the center of fluorescent sources (9.6 ± 5.4% error), as well as their transmural extent (15.7 ± 11.5% error). In ventricular slabs we were able to localize the sites of origin of intramural excitation waves with a precision of ± 1.6 mm. Transmural conduction velocities were, for the first time, measured optically from the surfaces and found to be 21.0 ± 12.4 cm/s. In conclusion, alternating transillumination is a promising technique for reliable reconstruction of depth and expansion rate of intramural activation wave fronts in cardiac tissue.

Recently, novel near-infrared (NIR) voltage-sensitive dyes were developed for imaging electrical activity in blood-perfused hearts and for tomographic applications. However, their usefulness for conventional surface mapping is unclear. The spectral shift to the NIR range significantly increases the penetration depth of light into the tissue, thus increasing the intramural volume contributing to the optical action potential (OAP). Here, we characterize both computationally and experimentally the effect of increased penetration depth on the OAP upstroke, the OAP component most sensitive to optical scattering and absorption, and the activation maps. Optical imaging of cardiac electrical activity was performed in isolated rat hearts (n = 5) paced from the LV mid free wall. We used the NIR dye JPW-6033 (excitation at 660nm, acquisition at >695nm). The conventional dye DI-4-ANEPPS (excitation at 532nm, acquisition at 700 DF50nm) was used for comparison. To simulate OAP we utilized a hybrid model that couples light transport equations with the model of electrical propagation. As expected, the switch from DI-4-ANEPPS to JPW-6033 significantly increased the upstroke duration: from 3.95 + or - 0.69ms to 5.39 + or - 0.82 ms, respectively. However, activation maps were largely unaffected. The correlation between the shape of the optical upstroke, and the averaged subsurface wave front orientation was also preserved. The computer simulations are in excellent agreement with the experimental data. In conclusion, our analysis suggests that despite significant increase in upstroke duration, the novel NIR dyes can be a useful alternative to conventional dyes in surface mapping applications.

Electrotonic interactions in cardiac tissue have been shown previously to modulate dynamical properties of the myocardium such as action potential duration (APD) and action potential duration restitution. A recent computational study indicated that these electrotonic effects may be strongest in small murine hearts. In the present study, we investigate experimentally how APD is modulated by activation sequence and pacing rate using optical mapping in Langendorff perfused rat hearts. Our results show that following an epicardial point stimulus, a strong correlation exists between epicardial APD and activation time, with decreasing APD for increasing activation time. This effect is preserved for all pacing frequencies (6-14 Hz) investigated in this study. Our experimental results are validated by detailed three-dimensional computer simulations. These simulations also demonstrate a strong transmural APD dependence on activation sequence, which, near the pacing site, is sufficient to mask the intrinsic transmural gradient.

Optical imaging allows mapping the complex spatiotemporal dynamics of transmembrane potential and intracellular calcium in cardiac tissue. Several studies have shown that the epi-fluorescent optical action potential contains contributions from the sub-epicardium, owing to scattering of photons in tissue. Hybrid electro-optical models have allowed careful quantification of these scattering effects and have lead to a better interpretation of the optical action potential. However, until now, these effects have not been investigated for optically recorded calcium transients. Here, we develop a hybrid model of cardiac dual calcium-voltage epi-fluorescence mapping. This model allows simulating both optical action potentials and optical calcium transients and investigating the effects of photon scattering on their synthesis. We find that optical calcium transients contain contributions from sub-epicardial layers up to 0.8 mm below the epicardium. These lead to significant differences in rise time and activation times between the optically acquired calcium signal and the epicardial intracellular calcium concentration. As has been the case with optically recording action potentials, these results should be taken into account in the interpretation of experimental optical measurements of intracellular calcium.