Timothy J. Cashman

Mitochondria play a central role in numerous physiological and pathological processes, and mitochondrial transplantation is emerging as a promising strategy to restore cellular function and mitigate disease. The success of this approach depends critically on the methods used to isolate, preserve, and retrieve intact, functional mitochondria. Objective: To optimize an isolation strategy that preserves mitochondrial integrity, dynamics, and metabolic activity and to evaluate conditions that enable short-term storage for future organelle biobanking applications. Methods: We compared a mitochondria isolation method developed in our laboratory (Protocol A) with a commercially available kit (Protocol B). Donor mitochondria were isolated from proximal tubular cells and transplanted into HEK293T recipient cells. Mitochondrial functionality was assessed following transfer into HEK293T cells by measuring reactive oxygen species (MitoSOX Red), oxygen consumption rate (OCR) using Seahorse XF analysis, and high-resolution imaging of mitochondrial morphology and dynamics. We further evaluated mitochondrial storage at low temperature and subsequent functional recovery. Results: Protocol A enabled faster isolation (~30 min) than Protocol B (~80 min) and yielded mitochondria with higher transplantation efficiency, greater OCR, preserved dynamic morphology, and lower oxidative stress. Mitochondria isolated using Protocol A remained metabolically active after transplantation and continued to exhibit fission and fusion, whereas those isolated using Protocol B showed reduced dynamic behavior. Importantly, mitochondria isolated with Protocol A retained functional integrity after low-temperature storage, supporting their potential for standardized preservation. Conclusions: This study presents a robust, efficient, and reproducible isolation and frozen-storage protocol that yields highly functional mitochondria suitable for transplantation. The ability to preserve mitochondrial function after storage further highlights the potential for developing organelle biobanks to support future research and therapeutic applications.

Cardiac excitation–contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral β-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

Rationale:: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. Objective:: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. Methods and Results:: Extending our previous hMSC–cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation–contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non–hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). Conclusions:: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The...