Computational design of custom therapeutic cells to correct failing human cardiomyocytes

Background: Myocardial delivery of non-excitable cells—namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)—remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes.Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte.Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics.Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells

Benchmarking mortality risk prediction from electrocardiograms

Several recent high-impact studies leverage large hospital-owned electrocardiographic (ECG) databases to model and predict patient mortality. MIMIC-IV, released September 2023, is the first comparable public dataset and includes 800,000 ECGs from a U.S. hospital system. Previously, the largest public ECG dataset was Code-15, containing 345,000 ECGs collected during routine care in Brazil. These datasets now provide an excellent resource for a broader audience to explore ECG survival modeling. Here, we benchmark survival model performance on Code-15 and MIMIC-IV with two neural network architectures, compare four deep survival modeling approaches to Cox regressions trained on classifier outputs, and evaluate performance at one to ten years. Our results yield AUROC and concordance scores comparable to past work (circa 0.8) and reasonable AUPRC scores (MIMIC-IV: 0.4-0.5, Code-15: 0.05-0.13) considering the fraction of ECG samples linked to a mortality (MIMIC-IV: 27\%, Code-15: 4\%). When evaluating models on the opposite dataset, AUROC and concordance values drop by 0.1-0.15, which may be due to cohort differences. All code and results are made public.Comment: 9 pages plus appendix, 2 figure

An Improved Reporter Identifies Ruxolitinib as a Potent and Cardioprotective CaMKII Inhibitor

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration–approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications

Abstract 130: Secretion of Angiogenic and Anti-apoptotic Factors Accompanies Mesenchymal Stem Cell-mediated Enhancement of Contractile Function in Engineered Cardiac Tissues

Previous studies using three-dimensional engineered cardiac tissues (ECT) demonstrated beneficial effects of mesenchymal stem cells (MSC) on contractile function, recapitulating findings in animal studies and in clinical trials. But the mechanisms by which MSC effect this functional enhancement remain unclear. This study tested the hypothesis that MSC-mediated enhancement of ECT function involves paracrine signaling, independent of direct cell-cell interaction with cardiomyocytes (CM). To create ECT, CM were isolated from neonatal rat ventricles, mixed with 2.0 mg/ml bovine type I collagen and 0.9 mg/ml Matrigel, and pipetted into a custom elastomeric mold with integrated force sensing end-posts. Two types of ECT were created: one containing 1.5 million CM (“CM-only”) and the other containing 1.5 million CM supplemented with 150,000 MSC (“MSC-CM hybrid”). Contractile function of CM-only ECT was then assessed during 2-Hz pacing either in the presence or absence of hybrid tissues sharing the same media. Media surrounding the tissues was also collected and analyzed via protein microarray (RayBiotech) combined with Ingenuity Pathway Analysis software. CM-only ECT cultured for 5 days showed aligned and compacted structure with a cross-sectional area of 0.39 ± 0.03 mm 2 and a twitch force of 4.7 ± 0.7 N (mean ± SD; n = 6). In CM-only tissues co-cultured for 5 days in shared media alongside hybrid tissues, cross-sectional area was unchanged (0.42 ± 0.02 mm 2 , p = 0.17, n=3), but twitch force increased two-fold to 9.8 ± 6.5 N (p = 0.08). Threshold voltage for electrical pacing of CM-only ECT also decreased from 0.54 ± 0.12 V/mm to 0.39 ± 0.07 V/mm when co-cultured with hybrid ECT (p = 0.097). Protein microarray analysis of the shared co-culture conditioned media showed enrichment (relative to CM-only conditioned media) of the angiogenic factors VEGF (1240x) and IL-1 (8.6x) as well as the cardiac anti-apoptotic/pro-survival factors TNF (194x), bFGF (55x), and bNGF (14.9x). In conclusion, these findings support paracrine signaling, independent of direct cell-cell contact, as one mechanism of MSC-mediated enhancement of CM function in rat ECT. Identification and isolation of defined cardiotropic factors may lead to novel molecular therapies for cardiac repair.</jats:p

Computational design of custom therapeutic cells to correct failing human cardiomyocytes

Background: Myocardial delivery of non-excitable cells—namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)—remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes.Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte.Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics.Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.</jats:p

Voltage-Sensor Transition Model for the Delayed Rectifier Current.

To model Idr, a modified version of Silverman’s two voltage-sensor transition Markovian-like model [33, 34] was used. As shown, there is one closed state (C1), one intermediate state (C2), and one open state (y). Each state has its own voltage-dependent rate functions (i.e., α, β, γ, and δ).</p

DataSheet1_Computational design of custom therapeutic cells to correct failing human cardiomyocytes.pdf

Background: Myocardial delivery of non-excitable cells—namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)—remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes.Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte.Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics.Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.</p

In silico Cell Therapy Model Restores Failing Human Myocyte Electrophysiology and Calcium Cycling in Fibrotic Myocardium

Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem cells (hMSCs), an emerging approach for treating the failing heart, has been limited by an incomplete understanding of the effects on host myocardium. This computational study aims to model hCIC and hMSC effects on electrophysiology and calcium cycling of healthy and diseased human cardiomyocytes (hCM), and reveals a possible cardiotherapeutic benefit independent of putative regeneration processes. First, we developed an original hCIC mathematical model with an electrical profile comprised of distinct experimentally identified ion currents. Next, we verified the model by confirming it is representative of published experiments on hCIC whole-cell electrophysiology and on hCIC co-cultures with rodent cardiomyocytes. We then used our model to compare electrophysiological effects of hCICs to other non-excitable cells, as well as clinically relevant hCIC-hMSC combination therapies and fused hCIC-hMSC CardioChimeras. Simulation of direct coupling of hCICs to healthy or failing hCMs through gap junctions led to greater increases in calcium cycling with lesser reductions in action potential duration (APD) compared with hMSCs. Combined coupling of hCICs and hMSCs to healthy or diseased hCMs led to intermediate effects on electrophysiology and calcium cycling compared to individually coupled hCICs or hMSCs. Fused hCIC-hMSC CardioChimeras decreased healthy and diseased hCM APD and calcium transient amplitude compared to individual or combined cell treatments. Finally, to provide a theoretical basis for optimizing cell-based therapies, we randomized populations of 2,500 models incorporating variable hMSC and hCIC interventions and simulated their effects on restoring diseased cardiomyocyte electrophysiology and calcium handling. The permutation simulation predicted the ability to correct abnormal properties of heart failure hCMs in fibrotic, but not non-fibrotic, myocardium. This permutation experiment also predicted paracrine signaling to be a necessary and sufficient mechanism for this correction, counteracting the fibrotic effects while also restoring arrhythmia-related metrics such as upstroke velocity and resting membrane potential. Altogether, our in silico findings suggest anti-fibrotic effects of paracrine signaling are critical to abrogating pathological cardiomyocyte electrophysiology and calcium cycling in fibrotic heart failure, and support further investigation of delivering an optimized cellular secretome as a potential strategy for improving heart failure therapy.</jats:p

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