Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes

<div><p>Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform—such as decreased action potential duration (APD) and plateau height—were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K⁺ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1 activity may offer increased safety as a cell delivery cardiotherapy at low levels of hMSC-hCM coupling.</p></div

hCM APD Sensitivity to hMSC Current Parameters.

<p>A sensitivity analysis based on 300 trials per cell type was performed to develop insight into the correlation between hCM APD and current parameters of (<b>A</b>) Type A hMSCs, (<b>B</b>) Type B hMSCs, and (<b>C</b>) Type C hMSCs, each at a 1:1 ratio with midcardial hCMs. hCM APD was most sensitive to Types A and B hMSC gap junctional conductance (G_junction) and delayed-rectifier current parameters (<i>G</i>_<i>dr</i>, <i>α</i>, <i>β</i>, <i>γ</i>, and <i>δ</i>). hCM APD was less sensitive to Type C hMSC G_junction, demonstrating that this cell type’s effects are less disruptive to the APD of coupled hCMs and caused less APD variability (i.e., lowest value of <i>σ</i>_APD, as noted in each panel). The normalized parameter sensitivity vector, <b>B</b>, was scaled by <i>σ</i>_APD to better illustrate the sensitivity of the APD output to each hMSC cell type.</p

Quantification of hCM Action Potential Waveform Following Direct hMSC-hCM Electrical Interactions.

<p>To further explore the effects of each family of hMSC, we quantified the relationship between (<b>A</b>) APD, (<b>B</b>) <i>V</i>_<i>APD</i>/2, (<b>C</b>) RMP, and the percentage of coupled (left panel) or fused (right panel) hMSCs in a homogeneously distributed hMSC-hCM population. hMSCs were coupled or fused to midcardial hCMs in an hMSC-hCM population ranging from 0% (control) to 80% hMSCs at 10% increments. These effects were compared to hCMs coupled to a passive R-C circuit-like hMSC cell. In general, Type C hMSCs resulted in the least variation in hCM APD and <i>V</i>_<i>APD</i>/2. The dotted lines represent the control condition of hCMs with no hMSC coupling or fusion.</p

Total Current Simulations of the Triad of hMSC Families.

<p>The whole-cell models developed in this study were validated by simulating <i>I</i>_tot,A, <i>I</i>_tot,B, and <i>I</i>_tot,C, as shown in (<b>A</b>), (<b>B</b>), and (<b>C</b>), respectively. Schematics of functional currents for each cell type are shown to the right of each simulation. The voltage protocol for each cell type is inset in (<b>C</b>). The simulations generally agree with the magnitude and behavior of representative experimental data [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.ref026" target="_blank">26</a>], and thus were used to predict the direct electrical interactions between hMSCs and hCMs.</p

VW Analysis on Cardiac Tissue With Randomly Inserted hMSCs.

<p>A VW analysis was performed to better understand the pro-arrhythmic potential of hMSC insertion in cardiac tissue. (<b>A</b>) Selected frames from a representative simulation of an S1–S2 interval of 380 ms that led to re-entry with 25% Type A hMSC random insertion into 2-D cardiac tissue. (<b>B</b>) The VWs for tissues with 0% (control), 5%, 15%, and 25% random insertion of hMSCs; at low levels of hMSC insertion, Type C hMSCs lead to the smallest increase in VW compared to control. (<b>C</b>) Analysis of S1–S2 intervals leading to conduction block (CB), re-entry (R), or uninterrupted propagation (UP) with 5% hMSC insertion. Error bars represent standard deviation based on three tissue configurations per condition.</p

<i>I</i>-<i>V</i> and Voltage Clamp Simulations of hMSC Currents.

<p>Comparison of experimental and fitted <i>I</i>-<i>V</i> curves for hMSC channels, and the resulting voltage clamp simulations. (<b>A</b>) Fitted <i>I</i>-<i>V</i> curve for I_KCa together with mean experimental data from Li et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.ref026" target="_blank">26</a>]. (<b>B</b>) Simulated voltage clamp experiment of I_KCa (voltage step protocol inset). (<b>C</b>) Theoretical <i>I</i>-<i>V</i> curve for I_dr and its fit to mean experimental data [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.ref026" target="_blank">26</a>]. (<b>D</b>) In silico voltage clamp experiment of I_dr (voltage step protocol shown in inset of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.g003" target="_blank">Fig 3C</a>). (<b>E</b>) A comparison of fitted theoretical and mean experimental <i>I</i>-<i>V</i> curve data [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.ref026" target="_blank">26</a>] for I_LCa. (<b>F</b>) Voltage clamp simulation for I_LCa (voltage step protocol inset). Comparisons between fitted theoretical and experimental <i>I</i>-<i>V</i> data [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005014#pcbi.1005014.ref026" target="_blank">26</a>] for I_to and I_Na are shown in (<b>G</b>) and (<b>I</b>), respectively. Voltage clamp simulations for I_to and I_Na are shown in (<b>H</b>) and (<b>J</b>), respectively (voltage step protocols inset).</p

hMSC Effects on Dispersion of Refractoriness and Restitution Curves.

<p>Effects of the types of hMSCs coupled, as well as the percentage of hMSCs coupled, on dispersion of refractoriness and restitution curves were examined. (<b>A</b>) APD dispersion (<i>ζ</i>) was lowest for Type C hMSCs. APD dispersion increased with higher percentages of coupled hMSCs. (<b>B</b>) APD restitution slopes, as well as the range of DIs for slopes greater than 1, decreased with hMSC percentage. Inset plot shows expanded region of APD restitution slopes greater than 1. (<b>C</b>) CV restitution curves were independent of hMSC type. As the percentage of inserted hMSCs increased, the maximum CV decreased.</p

Key hMSC Outward Currents Involved in Electrical Sinking.

<p>The main hMSC outward currents involved in a faster initiation of phases 3 and 4 of hCM action potentials were examined. The hMSC currents analyzed were: (<b>A</b>) I_KCa, (<b>B</b>) I_dr, and (<b>C</b>) I_to. During an hCM action potential, I_dr had the largest magnitude and area under the curve, which resulted in the greatest electrical sink effects.</p

hMSCs Act as Electrical Sources and Sinks in hCM APD Variations.

<p>hMSCs were coupled to midcardial hCMs in a 1:1 ratio to examine how hMSC membrane potential and gap current affected the hCM action potential. (<b>A</b>) Effects of coupling each type of hMSC on hCM action potentials, compared to hCM-only control. Phases 1 through 4 of the cardiac action potential are labeled for reference. (<b>B</b>) hMSC transmembrane voltage throughout an hCM action potential. (<b>C</b>) The resulting gap currents betwen hMSCs and hCMs due to differences in voltage between cell types. I_Gap was defined as current flowing from the hMSC to the hCM (i.e., I_Gap,hCM).</p