Heart failure remains a leading cause of death globally, despite advances in treatment. Stem cell therapy offers a potential solution, with heart explant-derived cells (EDCs) emerging as a promising candidate for paracrine-based therapy. EDCs, specifically the CD105⁺ population, have demonstrated effectiveness in reducing pathological cardiac remodeling and improving myocardial function. However, the clinical efficacy of EDCs can be affected by patient comorbidities, and identifying reliable markers to predict therapeutic potential remains a challenge. This study focuses on the regulation of membrane potential (*V*_mem) and intracellular Ca²⁺, crucial factors influencing stem cell properties. Previous research showed that the intermediate-conductance Ca²⁺-activated K⁺ channel KCa3.1 (encoded by the *KCNN4* gene) is critical for the function of bone-marrow-derived mesenchymal stem cells and resident cardiac c-Kit⁺ cells. KCa3.1 channel opening hyperpolarizes the cell membrane, increasing the driving force for Ca²⁺ entry and enhancing transmembrane Ca²⁺ flux. This study hypothesizes that KCa3.1 channel activity influences therapeutically relevant cells and that *KCNN4* overexpression optimizes *V*_mem during store-operated Ca²⁺ entry (SOCE), thereby improving therapeutic efficacy. The research aims to test this hypothesis using *ex vivo* expanded human heart cells in an established immunodeficient mouse model of ischemic cardiomyopathy.

The literature review highlights the significant global burden of heart failure and the emergence of stem cell therapy as a promising treatment approach. Several studies emphasize the therapeutic potential of EDCs, particularly their paracrine effects in reducing cardiac remodeling and improving myocardial function. However, the clinical translation of EDC therapy has been hampered by inconsistencies in treatment outcomes influenced by patient comorbidities. Previous research by the authors and others established the crucial role of KCa3.1 channels in regulating the function of other progenitor cell types, highlighting the potential link between ion channel activity and cell therapeutic potential. This study builds upon these findings by exploring the specific role of KCa3.1 channels in EDC function and therapeutic efficacy.

Human EDCs were obtained from left atrial appendages of patients undergoing heart surgery. The study characterized endogenous Ca²⁺-activated K⁺ channels in CD90⁺ and CD90⁻ EDC subpopulations using patch-clamp techniques. Electrophysiological properties, including reversal potential and current density, were assessed under various conditions, including the application of KCa channel blockers (paxilline and TRAM-34). Store-operated Ca²⁺ entry (SOCE) was induced, and its effect on *V*_mem was monitored. Lentiviral transduction was used to overexpress *KCNN4* in EDCs, and the effects on *I*_KCa3.1, *V*_mem, and intracellular Ca²⁺ were measured using patch-clamp and Fluo-4 calcium imaging. In vitro studies assessed the impact of *KCNN4* overexpression on EDC proliferation and apoptosis. In vivo experiments utilized a NOD/SCID IL2Ry mouse model of ischemic cardiomyopathy. Mice underwent left coronary artery ligation, followed by intramyocardial injection of EV-EDCs, KCNN4-EDCs, or NT-EDCs. Cardiac function was assessed using echocardiography and invasive hemodynamics. Histological analyses were performed to evaluate infarct size, wall thickness, neovascularization, and cell retention. Cytokine and miRNA profiles were examined using proteomic arrays and Nanostring technology, respectively. Extracellular vesicle production was characterized using Nanoparticle Tracking Analysis. Statistical analysis employed appropriate tests for repeated and non-repeated measures, and p<0.05 was considered statistically significant. Animal procedures were approved by the University of Ottawa Animal Care Committee.

The study revealed distinct endogenous currents in human EDCs, with CD90⁻ cells exhibiting significantly more negative reversal potentials than CD90⁺ cells. Two types of Ca²⁺-dependent K⁺ currents were identified: *I*_BKCa and *I*_KCa3.1. *V*_mem in CD90⁻ cells was found to be largely determined by TRAM-34-sensitive KCa3.1 conductance, which was absent in CD90⁺ cells. SOCE induced a strong hyperpolarization in CD90⁻ cells, mediated by *I*_KCa3.1. *KCNN4* gene transfer significantly increased *KCNN4* expression, leading to membrane hyperpolarization, increased intracellular Ca²⁺, and enhanced proliferation. In the murine model, KCNN4-transferred EDCs demonstrated significantly improved cardiac function, smaller infarct size, increased infarct wall thickness, preserved viable tissue, and enhanced peri-infarct neovascularization compared to control groups. Increased retention of transplanted cells was also observed. *KCNN4* overexpression increased the production of cytokines involved in angiogenesis, post-infarct healing, and immunomodulation. It also increased extracellular vesicle production and altered the miRNA expression profile, further contributing to the improved healing-promoting signature of the EDC secretome. Importantly, only the recombined KCNN4 cells (i.e., KCNN4 CD90⁻ and KCNN4 CD90⁺) significantly improved cell-treatment outcomes, suggesting synergistic effects within the mixed cell population. There was no detectable effect of EDC transplantation on cardiac electrophysiology in vivo.

The findings demonstrate that the therapeutic potential of human heart-derived cells is significantly influenced by the expression and function of the KCa3.1 channel. The selective expression of KCa3.1 in the therapeutically active CD90⁻ EDC subpopulation highlights its importance in cell function and therapeutic efficacy. Genetic manipulation to enhance KCa3.1 channel expression improved both the proliferation and paracrine properties of EDCs, leading to superior cardiac repair in the mouse model. The observed improvements in cardiac function are likely mediated through increased cytokine and nanoparticle secretion, stimulating endogenous repair mechanisms. This study is groundbreaking in demonstrating that manipulating plasma membrane ion channels can significantly enhance the therapeutic effects of progenitor cells, offering a novel approach to improve the clinical outcomes of cardiac cell therapy.

This study provides compelling evidence that enhancing KCa3.1 channel function via *KCNN4* overexpression significantly improves the therapeutic efficacy of heart-derived cells in a murine model of ischemic cardiomyopathy. This approach offers a novel strategy for improving the clinical outcomes of cardiac cell therapy. Future research should focus on optimizing gene delivery methods for clinical translation and investigating the detailed molecular mechanisms underlying the beneficial effects of KCa3.1 channel modulation. Exploring the generalizability of this approach to other progenitor cell types is also warranted.

The study used an immunodeficient mouse model, which may not fully replicate the immune response in humans. The use of lentiviral vectors for gene transfer raises potential safety concerns related to insertional mutagenesis, requiring further investigation for clinical translation. The time point of cell injection (1 week post-ligation) might not fully reflect the complex processes of scar formation and remodeling in later stages of post-infarct healing. Further investigations are required to explore other cell populations and mechanisms involved in the beneficial effects of KCNN4 overexpression.