Background: Cholesterol accumulation and calcium depletion induce hepatic injury via the endoplasmic reticulum (ER) stress response. ER stress regulates the calcium imbalance between the ER and mitochondria. We previously reported that phosphatase of regenerating liver-1 (PRL-1)-overexpressing placenta-derived mesenchymal stem cells (PD-MSCs^PRL−1) promoted liver regeneration via mitochondrial dynamics in a cirrhotic rat model. However, the role of PRL-1 in ER stress-dependent calcium is not clear. Therefore, we demonstrated that PD-MSCs^PRL−1 improved hepatic functions by regulating ER stress and calcium channels in a rat model of bile duct ligation (BDL). Methods: Liver cirrhosis was induced in Sprague–Dawley (SD) rats using surgically induced BDL for 10 days. PD-MSCs and PD-MSCs^PRL−1 (2 × 10⁶ cells) were intravenously administered to animals, and their therapeutic effects were analyzed. WB-F344 cells exposed to thapsigargin (TG) were cocultured with PD-MSCs or PD-MSCs^PRL−1. Results: ER stress markers, e.g., eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were increased in the nontransplantation group (NTx) compared to the control group. PD-MSCs^PRL−1 significantly decreased ER stress markers compared to NTx and induced dynamic changes in calcium channel markers, e.g., sarco/endoplasmic reticulum Ca²⁺ -ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3R), mitochondrial calcium uniporter (MCU), and voltage-dependent anion channel 1 (VDAC1) (*p < 0.05). Cocultivation of TG-treated WB-F344 cells with PD-MSCs^PRL−1 decreased cytosolic calmodulin (CaM) expression and cytosolic and mitochondrial Ca²⁺ concentrations. However, the ER Ca²⁺ concentration was increased compared to PD-MSCs (*p  < 0.05). PRL-1 activated phosphatidylinositol-3-kinase (PI3K) signaling via epidermal growth factor receptor (EGFR), which resulted in calcium increase via CaM expression. Conclusions: These findings suggest that PD-MSCs^PRL−1 improved hepatic functions via calcium changes and attenuated ER stress in a BDL-injured rat model. Therefore, these results provide useful data for the development of next-generation MSC-based stem cell therapy for regenerative medicine in chronic liver disease.

Retinal degenerative diseases result from oxidative stress and mitochondrial dysfunction, leading to the loss of visual acuity. Damaged retinal pigment epithelial (RPE) and photoreceptor cells undergo mitophagy. Pigment epithelium-derived factor (PEDF) protects from oxidative stress in RPE and improves mitochondrial functions. Overexpression of PEDF in placenta-derived mesenchymal stem cells (PD-MSCs; PD-MSCs^PEDF) provides therapeutic effects in retinal degenerative diseases. Here, we investigated whether PD-MSCs^PEDF restored the visual cycle through a mitophagic mechanism in RPE cells in hydrogen peroxide (H₂O₂)-injured rat retinas. Compared with naïve PD-MSCs, PD-MSCs^PEDF augmented mitochondrial biogenesis and translation markers as well as mitochondrial respiratory states. In the H₂O₂-injured rat model, intravitreal administration of PD-MSCs^PEDF restored total retinal layer thickness compared to that of naïve PD-MSCs. In particular, PTEN-induced kinase 1 (PINK1), which is the major mitophagy marker, exhibited increased expression in retinal layers and RPE cells after PD-MSC^PEDF transplantation. Similarly, expression of the visual cycle enzyme retinol dehydrogenase 11 (RDH11) showed the same patterns as PINK1 levels, resulting in improved visual activity. Taken together, these findings suggest that PD-MSCs^PEDF facilitate mitophagy and restore the loss of visual cycles in H₂O₂-injured rat retinas and RPE cells. These data indicate a new strategy for next-generation MSC-based treatment of retinal degenerative diseases.

Mesenchymal stem cell (MSC) therapy in chronic liver disease is associated with mitochondrial anaerobic metabolism. Phosphatase of regenerating liver-1 (PRL-1), known as protein tyrosine phosphatase type 4A, member 1 (PTP4A1), plays a critical role in liver regeneration. However, its therapeutic mechanism remains obscure. The aim of this study was to establish genetically modified bone marrow (BM)-MSCs overexpressing PRL-1 (BM-MSCs^PRL-1) and to investigate their therapeutic effects on mitochondrial anaerobic metabolism in a bile duct ligation (BDL)-injured cholestatic rat model. BM-MSCs^PRL-1 were generated with lentiviral and nonviral gene delivery systems and characterized. Compared with naive cells, BM-MSCs^PRL-1 showed an improved antioxidant capacity and mitochondrial dynamics and decreased cellular senescence. In particular, mitochondrial respiration in BM-MSCs^PRL-1 generated using the nonviral system was significantly increased as well as mtDNA copy number and total ATP production. Moreover, transplantation of BM-MSCs^PRL-1 generated using the nonviral system had predominantly antifibrotic effects and restored hepatic function in a BDL rat model. Decreased cytoplasmic lactate and increased mitochondrial lactate upon the administration of BM-MSCs^PRL-1 indicated significant alterations in mtDNA copy number and ATP production, activating anaerobic metabolism. In conclusion, BM-MSCs^PRL-1 generated by a nonviral gene delivery system enhanced anaerobic mitochondrial metabolism in a cholestatic rat model, improving hepatic function.

The cardiac sodium–calcium exchanger (NCX1) is a critical regulator of intracellular calcium (Ca) in cardiomyocytes. Although strongly regulated by pH, the role of NCX1 pH sensitivity in cardiac function and intracellular Ca regulation is not understood. We used CRISPR/Cas9 to produce a pH-resistant NCX1 mouse by replacing histidine 165 with alanine (H165A). H165A mice live into adulthood and are fertile. Comparative studies in isolated ventricular myocytes demonstrated that NCX1 current (I _NCX ) was significantly inhibited by lowering pH _i to ~6.5 in the wild type (WT) but was unaffected in myocytes from pH-resistant H165A mice. Similarly, in WT cardiomyocytes loaded with the Ca indicator fura-2 AM, Ca transient amplitude increased dramatically during field stimulation when pH _i was lowered to 6.5, consistent with impaired Ca efflux caused by proton-mediated NCX1 inhibition. In contrast, Ca transients were unchanged by low pH _i in H165A myocytes, indicating effective Ca efflux by the mutant exchanger despite acidosis. When we subjected H165A mouse hearts to ischemia/reperfusion, where cellular injury is thought to result in part from Ca accumulation and inhibition of NCX1 by ischemic acidosis, there was reduced injury and improved recovery of function compared to WT. These results reveal that maintaining NCX1 activity during ischemia, and presumably maintaining Ca extrusion, is a powerful approach to protect or precondition the heart against injury that would otherwise occur subsequently during reperfusion. We conclude that allosteric regulation of NCX1 by protons is a highly relevant physiological process that takes place in clinical settings associated with abnormally low pH, such as cardiac ischemia.

BACKGROUND: In addition to lethal ventricular arrhythmias, arrhythmogenic cardiomyopathy (ACM) is associated with conduction abnormalities, bradycardias, and reduced expression of the scaffolding junctional protein zonula occludens-1 (ZO-1). Reduced ZO-1 expression is also seen in dilated cardiomyopathy, which is far more common than ACM. Conduction abnormalities are likewise a feature of ZO-1 cardiac-specific knockout (ZO-1cKO) mice. However, the role of ZO-1 in sinoatrial node (SAN) automaticity has not been studied. OBJECTIVE: To investigate the role of ZO-1 in SAN automaticity and elucidate the mechanisms by which ZO-1 deficiency leads to SAN dysfunction. METHODS: ZO-1 cardiac-specific knockout (ZO-1cKO) mice were generated by crossing ZO-1 floxed mice with αMHC-nuclear Cre mice. SAN/atrial tissue and isolated SAN cells were examined using optical mapping, single-cell patch clamp, and quantitative PCR techniques to assess functional alterations caused by ZO-1 loss. RESULTS: ZO-1cKO mice exhibited enlarged atria and SAN area compared to control mice, with normal left ventricular function. Electrocardiograms showed sinus bradycardia, sinus pauses and atrioventricular block. Optical mapping revealed a caudal shift in the SAN leading region and reduced intra-atrial conduction velocity in ZO-1cKO mice. Patch-clamp recordings from isolated SAN cells showed reduced spontaneous action potential frequency and diastolic depolarization rate, while voltage-clamp revealed a marked reduction in pacemaker current (I _f ). CONCLUSION: ZO-1 expression is essential for SAN automaticity. Its loss impairs SAN impulse generation by reducing pacemaker current and hampering atrial conduction, leading to bradyarrhythmia, conduction delay and block. These findings help explain impulse generation and conduction abnormalities in ACM and other cardiomyopathies.