Structural and Functional Determinants of Cardiac Impulse Propagation and Arrhythmias.

Each year, sudden cardiac death (SCD) attributed to ventricular fibrillation (VF) kills approximately 200,000 people in the United States. However, the mechanisms responsible for VF, and therefore VF-related SCD, are incompletely understood. My PhD studies focused on two major topics directly related to the mechanisms of reentry in VF. My general approach was based on the use of neonatal cardiac cell monolayers, gene transfer, immunolocalization, patch clamping and optical mapping techniques. First, I examined how a delayed rectifier potassium channel gene (hERG) involved in cardiac repolarization affects reentry frequency in a ventricular myocyte monolayer model of reentry. The results provided strong evidence for a role of hERG in controlling the frequency and stability of reentry. The mechanisms underlying the acceleration in reentry frequency were shown to be action potential duration (APD) shortening and a transient hyperpolarization after each action potential. APD shortening reduced reentry wavelength which prevented wave front-wave tail interactions and increased reentry stability. The transient hyperpolarization enhanced sodium channel availability and excitability of tissue ahead of the propagating electrical wave front. Together they set the stage for fast and stable reentry that maintains VF. Second, I examined the principle of whether rescuing normal electrical impulse propagation in damaged or fibrotic myocardium using cell therapy would be an effective approach to alter reentry behavior. Electrically excitable cardiac fibroblasts were generated using viral constructs encoding Kir2.1, NaV1.5 and Cx43 proteins. Excitable fibroblasts were able to form monolayers and conduct electrical waves at high velocity. When used to replace normal fibroblasts in heterocellular monolayers, they significantly increased conduction velocity to values similar to those of pure myocytes monolayers. Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks. Altogether, the work accomplished in my dissertation should lead to a better understanding of VF and to the development of novel therapeutic approaches for the prevention of SCD.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/98057/1/houl_1.pd

A fully-automated low-cost cardiac monolayer optical mapping robot.

Scalable and high-throughput electrophysiological measurement systems are necessary to accelerate the elucidation of cardiac diseases in drug development. Optical mapping is the primary method of simultaneously measuring several key electrophysiological parameters, such as action potentials, intracellular free calcium and conduction velocity, at high spatiotemporal resolution. This tool has been applied to isolated whole-hearts, whole-hearts in-vivo, tissue-slices and cardiac monolayers/tissue-constructs. Although optical mapping of all of these substrates have contributed to our understanding of ion-channels and fibrillation dynamics, cardiac monolayers/tissue-constructs are scalable macroscopic substrates that are particularly amenable to high-throughput interrogation. Here, we describe and validate a scalable and fully-automated monolayer optical mapping robot that requires no human intervention and with reasonable costs. As a proof-of-principle demonstration, we performed parallelized macroscopic optical mapping of calcium dynamics in the well-established neonatal-rat-ventricular-myocyte monolayer plated on standard 35 mm dishes. Given the advancements in regenerative and personalized medicine, we also performed parallelized macroscopic optical mapping of voltage dynamics in human pluripotent stem cell-derived cardiomyocyte monolayers using a genetically encoded voltage indictor and a commonly-used voltage sensitive dye to demonstrate the versatility of our system.The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). The study was supported by the Ministry of Science and Innovation (MCIN) (PID2019-109329RB-I00), the Fundación Interhospitalaria para la Investigación Cardiovascular, the McEwen Stem Cell Institute, the Canada Research Chairs Program, the Stem Cell Network, the University of Toronto’s Medicine by Design/Canada First Research Excellence Fund initiative, and Ted Rogers Centre for Heart Research Education Fund.S

A fully-automated low-cost cardiac monolayer optical mapping robot

Scalable and high-throughput electrophysiological measurement systems are necessary to accelerate the elucidation of cardiac diseases in drug development. Optical mapping is the primary method of simultaneously measuring several key electrophysiological parameters, such as action potentials, intracellular free calcium and conduction velocity, at high spatiotemporal resolution. This tool has been applied to isolated whole-hearts, whole-hearts in-vivo, tissue-slices and cardiac monolayers/tissue-constructs. Although optical mapping of all of these substrates have contributed to our understanding of ion-channels and fibrillation dynamics, cardiac monolayers/tissue-constructs are scalable macroscopic substrates that are particularly amenable to high-throughput interrogation. Here, we describe and validate a scalable and fully-automated monolayer optical mapping robot that requires no human intervention and with reasonable costs. As a proof-of-principle demonstration, we performed parallelized macroscopic optical mapping of calcium dynamics in the well-established neonatal-rat-ventricular-myocyte monolayer plated on standard 35 mm dishes. Given the advancements in regenerative and personalized medicine, we also performed parallelized macroscopic optical mapping of voltage dynamics in human pluripotent stem cell-derived cardiomyocyte monolayers using a genetically encoded voltage indictor and a commonly-used voltage sensitive dye to demonstrate the versatility of our system

Genetically Engineered Excitable Cardiac Myofibroblasts Coupled to Cardiomyocytes Rescue Normal Propagation and Reduce Arrhythmia Complexity in Heterocellular Monolayers

<div><h3>Rationale and Objective</h3><p>The use of genetic engineering of unexcitable cells to enable expression of gap junctions and inward rectifier potassium channels has suggested that cell therapies aimed at establishing electrical coupling of unexcitable donor cells to host cardiomyocytes may be arrhythmogenic. Whether similar considerations apply when the donor cells are electrically excitable has not been investigated. Here we tested the hypothesis that adenoviral transfer of genes coding Kir2.1 (I_K1), Na_V1.5 (I_Na) and connexin-43 (Cx43) proteins into neonatal rat ventricular myofibroblasts (NRVF) will convert them into fully excitable cells, rescue rapid conduction velocity (CV) and reduce the incidence of complex reentry arrhythmias in an <em>in vitro</em> model.</p> <h3>Methods and Results</h3><p>We used adenoviral (Ad-) constructs encoding Kir2.1, Na_V1.5 and Cx43 in NRVF. In single NRVF, Ad-Kir2.1 or Ad-Na_V1.5 infection enabled us to regulate the densities of I_K1 and I_Na, respectively. At varying MOI ratios of 10/10, 5/10 and 5/20, NRVF co-infected with Ad-Kir2.1+ Na_V1.5 were hyperpolarized and generated action potentials (APs) with upstroke velocities >100 V/s. However, when forming monolayers only the addition of Ad-Cx43 made the excitable NRVF capable of conducting electrical impulses (CV = 20.71±0.79 cm/s). When genetically engineered excitable NRVF overexpressing Kir2.1, Na_V1.5 and Cx43 were used to replace normal NRVF in heterocellular monolayers that included neonatal rat ventricular myocytes (NRVM), CV was significantly increased (27.59±0.76 cm/s vs. 21.18±0.65 cm/s, p<0.05), reaching values similar to those of pure myocytes monolayers (27.27±0.72 cm/s). Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks in heterocellular monolayers formed by excitable compared with unexcitable NRVF.</p> <h3>Conclusion</h3><p>Viral transfer of genes coding Kir2.1, Na_V1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate APs. The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential.</p> </div

K/Na/Cx43 NRVF rescued normal conduction velocity. A.

<p>Activation maps from monolayers of myocytes only (M, left), uninfected NRVF/NRVM co-culture (UI Fb/M, middle), and K/Na/Cx43 NRVF/NRVM co-culture (K/Na/Cx43 Fb/M, right). <b>B.</b> Quantification of conduction velocities at varies pacing cycle lengths in monolayers of myocytes (filled circles), UI Fb/M (open circle), and K/Na/Cx43 Fb/M (filled square).</p

Action potential propagation in 2D monolayer of K/Na/Cx43 NRVF.

<p><b>A.</b> Representative activation map of action potential propagation (1Hz pacing) in K/Na/Cx43 NRVF monolayer. Scale bar = 10 mm. <b>B.</b> Conduction velocity measured at different pacing cycle lengths from 1000 ms to 200 ms (n = 7). <b>C.</b> APD₇₅ and APD₅₀ measured at pacing cycle lengths from 1000 ms to 200 ms (n = 7).</p

Adenoviral expressions of Cx43 in NRVF after 48 hours of infection.

<p><b>A.</b> Immunostaining of control and Ad-Cx43 infected NRVF using an antibody for Cx43 (green) showed increased expression of Cx43 on cell membrane. Scale bar = 50 µm <b>B.</b> Western blot showed a 30-fold increase in the total amount of Cx43 proteins in Ad-Cx43 infected myofibroblasts compared with control uninfected NRVF. <b>C.</b> FRAP experiments showed strong functional coupling between Ad-Cx43 infected NRVF. Red circles indicated the target cells that were photobleached. Green circles showed the possible donor cells next to target cells. Blue and yellow circles were used to evaluate the background fluorescence. <b>D.</b> Quantification of the florescence recovery within six minutes after photobleaching in control (open circles) and Ad-Cx43 infected (filled circles) NRVF.</p

K/Na/Cx43 NRVF increased reentry frequency and reduced wavebreaks. A.

<p>Snapshots (top) and Time-Space Plot (bottom) from representative optical mapping movies in monolayers of myocytes (left), UI Fb/M (middle), and K/Na/Cx43 Fb/M (right). <b>B.</b> Quantification of rotation frequencies in monolayers of myocytes (filled circle), UI Fb/M (open circle), and K/Na/Cx43 Fb/M (filled square). <b>C.</b> Quantification of phase singularities in monolayers of myocytes (filled circle), UI Fb/M (open circle), and K/Na/Cx43 Fb/M (filled square).</p

Adenoviral expressions of Kir2.1 and Na_V1.5 proteins in NRVF after 48 hours of infection.

<p><b>A.</b> GFP-tagged Kir2.1 channel expression. Left: fluorescent micrograph of a passage 3 NRVF 48 h after Ad-Kir2.1 infection. Right: corresponding phase contrast micrograph. Scale bar = 100 µm. <b>B.</b> Voltage clamp protocol (top right) and representative example of currents (top left) from Ad-Kir2.1 infected NRVF; and I–V relationship (bottom) of the BaCl₂ sensitive currents normalized to cell capacitance in NRVF infected with Ad-Kir2.1 at 5 MOI (open circles) and 10 MOI (filled circles). <b>C.</b> Voltage clamp protocol (top right) and representative example of currents from Ad-Na_V1.5 (top left) infected NRVF; and I–V relationship (bottom) of I_Na normalized to cell capacitance in NRVF infected with Ad-Na_V1.5 at 10 MOI (filled circles) and 20 MOI (open circles). <b>D.</b> Normalized activation and inactivation curves of I_Na. <b>E.</b> Normalized recovery from inactivation curve of I_Na. The curves in D and E are not different statistically. *: p<0.05.</p

Cell size of NRVF and NRVM.

<p>Cell size of NRVF and NRVM.</p