Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells

Clinical trials have shown that electric stimulation (ELSM) using either cardiac resynchronization therapy (CRT) or cardiac contractility modulation (CCM) approaches is an effective treatment for patients with moderate to severe heart failure, but the mechanisms are incompletely understood. Extracellular vesicles (EV) produced by cardiac mesenchymal stem cells (C-MSC) have been reported to be cardioprotective through cell-to-cell communication. In this study, we investigated the effects of ELSM stimulation on EV secretion from C-MSCs (C-MSCELSM). We observed enhanced EV-dependent cardioprotection conferred by conditioned medium (CM) from C-MSCELSM compared to that from non-stimulated control C-MSC (C-MSCCtrl). To investigate the mechanisms of ELSM-stimulated EV secretion, we examined the protein levels of neutral sphingomyelinase 2 (nSMase2), a key enzyme of the endosomal sorting complex required for EV biosynthesis. We detected a time-dependent increase in nSMase2 protein levels in C-MSCELSM compared to C-MSCCtrl. Knockdown of nSMase2 in C-MSC by siRNA significantly reduced EV secretion in C-MSCELSM and attenuated the cardioprotective effect of CM from C-MSCELSM in HL-1 cells. Taken together, our results suggest that ELSM-mediated increases in EV secretion from C-MSC enhance the cardioprotective effects of C-MSC through an EV-dependent mechanism involving nSMase2.


Introduction
Cardiac mesenchymal stem cells (C-MSCs) reside in the heart and express the early cardiac-specific transcription factor GATA4 and MSC markers, including Sca-1, CD105, and CD140 [1][2][3]. These C-MSCs have cardiac reparative properties via a paracrine mechanism mediated by angiogenic and other factors [1,4]. Extracellular vesicles (EVs) are common membrane-bound vesicles that contain various biomolecules, such as lipids, proteins, and nucleic acids. Exosomes are a specific class of small EVs (sEVs), <200 nm in size, that participate in intercellular communication and can be secreted by MSCs [5]. EVs are produced from cells by exocytosis and are taken up by target cells, thus transmitting biological signals between cells locally or distantly [6]. We previously reported that C-MSCderived EVs protected the acutely ischemic heart from reperfusion injury [7]. Moreover, transplantation of C-MSC-derived EVs increased cardiomyocyte proliferation and enhanced cardiac angiogenesis in a mouse model of myocardial infarction (MI) [3]. A recent metaanalysis of pre-clinical animal studies using stem cell-derived EVs suggested that they are effective at eliciting cardiac repair post-MI [8].
Landmark trials such as COMPANION have shown that cardiac-resynchronization therapy (CRT) improves cardiac function in patients with advanced chronic heart failure [9].

Electron Microscopy and Zeta Analysis
For transmission electron microscopy (TEM) morphology evaluation, 3 µL of EV pellet was placed on formvar carbon-coated 200-mesh copper electron microscopy grids and incubated for 5 min at room temperature (RT), followed by standard uranyl acetate staining. The grid was washed with three aliquots of PBS and allowed to become semi-dry at room temperature before observation with a transmission electron microscope (JEOL JEM 1230, Peabody, MA, USA).
We measured the size and concentration of EV in CM with nanoparticle tracking analysis (NTA) using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and the corresponding software, ZetaView 8.02.28. The ZetaView system was calibrated using 100 nm polystyrene particles.

Small-Interference RNA (siRNA) Transfection
Mouse C-MSC in 6-well plates were incubated with 40 nM of mouse nSMase2 siRNA or scrambled negative control (NC) siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Lipofectamine RNAiMax transfection reagent (Thermo Fisher, Waltham, MA, USA) in 2 mL medium for 48 h at 37 • C.

Assessment of Acetylcholinesterase (AchE) Activity
The concentration of EVs was quantified by measuring the activity of AchE (a typical EV marker) using the Amplex ® Red Acetylcholine/Acetylcholinesterase Assay Kit (Thermo Fisher) as previously described [23]. Briefly, 10 µL EV fractions were suspended in 90 µL of PBS. Then, 100 µL of diluted EVs were added to each well of a 96-well flat-bottomed microplate. Next, the EV samples were mixed with 100 µL of the working solution, including Amplex Red reagent containing HRP, choline oxidase, and acetylcholinesterase. AchE activity was measured using the GloMax Discover System (Promega, Madison, WI, USA) after incubation in the dark at room temperature for 30 min. For each well, the relative AChE activity was calculated by subtracting background fluorescence, normalizing to protein concentration, and then normalizing to the Ctrl si-NC group.

Cell Apoptosis Assay
We used an in vitro hypoxia model to simulate cardiac ischemia. Mouse cardiomyocyte cells (HL-1) cultured in BD GasPak Pouches (BD Biosciences, Cockeysville, MD, USA) were subjected to hypoxia (0.1% oxygen) for 16 h [25]. HL-1 cell apoptosis was detected using the DeadEnd™ Colorimetric TUNEL System. The TUNEL assay was performed according to the manufacturer's instructions with minor modifications. The TUNEL positivity of cells was quantified by fluorescence-activated cell sorting (FACS) analysis using a NovoCyte Quanteon flow cytometer (Agilent, Santa Clara, CA, USA). We first compared the apoptosis of hypoxia-exposed HL-1 cells incubated with CM from electrically stimulated C-MSC (C-MSC ELSM ) or control C-MSC (C-MSC Ctrl ). We also compare the apoptosis of hypoxiaexposed HL-1 cells incubated with EV-depleted (EV-del) CM from C-MSC ELSM;EV-del and C-MSC Ctrl;EV-del . To interrogate the functional role of nSMase2 in EV-mediated cytoprotection, we compared apoptosis in HL-1 cells incubated with CM from C-MSC treated with siRNA to knock down nSMase2 versus control siRNA.

Statistical Analysis
All data were expressed as the mean ± SEM of at least three independent experiments for each group of different biological samples. An unpaired student's t-test (GraphPad Prism version 9.41) was used to compare two groups. One-way ANOVA followed by Tukey's post-hoc tests was used for comparisons between more than two experimental groups. Values of p < 0.05 were considered statistically significant.

ELSM Stimulates EV Secretion of C-MSC
Morphological analysis (electron microscopy) of pellets collected from conditioned medium of C-MSC treated without and with ELSM showed the typical appearance of extracellular vesicles ( Figure 1A,B). The size of EVs was determined using ZetaView ® , with EVs from C-MSC Ctrl consisting of particles with an average diameter of 123.0 ± 65 nm, whereas EVs from C-MSC ELSM had an average diameter of 148.8 ± 72 nm. The concentration of EVs in CM from C-MSC Ctrl was 2.1 × 10 9 particles/Ml, whereas the concentration of EVs from C-MSC ELSM was 2.9 × 10 9 particles/mL, indicating that ELSM treatment increased EV secretion from C-MSC by approximately 38% (Figure 1C,D). medium of C-MSC treated without and with ELSM showed the typical appearance of extracellular vesicles ( Figure 1A,B). The size of EVs was determined using ZetaView ® , with EVs from C-MSC Ctrl consisting of particles with an average diameter of 123.0 ± 65 nm, whereas EVs from C-MSC ELSM had an average diameter of 148.8 ± 72 nm. The concentration of EVs in CM from C-MSC Ctrl was 2.1 × 10 9 particles/Ml, whereas the concentration of EVs from C-MSC ELSM was 2.9 × 10 9 particles/mL, indicating that ELSM treatment increased EV secretion from C-MSC by approximately 38% (Figure 1C,D).

EVs from ELSM-Treated C-MSC Protect Cardiomyocytes against Hypoxia-Induced Apoptosis
We previously reported that C-MSC-derived EVs protected cardiomyocytes from acute myocardial ischemia/reperfusion injury [7]. To investigate whether the ELSM-induced increase in EV secretion might enhance the cardioprotective effects, we incubated HL-1 cardiomyocytes with CM from C-MSC ELSM or C-MSC Ctrl under hypoxic stress for 16 h. The TUNEL assay showed significantly reduced apoptosis in HL-1 cells treated with CM from MSC ELSM as compared with C-MSC Ctrl (Figure 2A,B,E). However, when we treated HL-1 cardiomyocytes with EV-depleted CM, the differences in apoptosis between

EVs from ELSM-Treated C-MSC Protect Cardiomyocytes against Hypoxia-Induced Apoptosis
We previously reported that C-MSC-derived EVs protected cardiomyocytes from acute myocardial ischemia/reperfusion injury [7]. To investigate whether the ELSM-induced increase in EV secretion might enhance the cardioprotective effects, we incubated HL-1 cardiomyocytes with CM from C-MSC ELSM or C-MSC Ctrl under hypoxic stress for 16 h. The TUNEL assay showed significantly reduced apoptosis in HL-1 cells treated with CM from MSC ELSM as compared with C-MSC Ctrl (Figure 2A,B,E). However, when we treated HL-1 cardiomyocytes with EV-depleted CM, the differences in apoptosis between MSC ELSM and C-MSC Ctrl were abolished, suggesting that the ELSM-induced, enhanced cardioprotective effect was EV dependent ( Figure 2C-E).

ELSM Increases nSMase2 Expression in C-MSC
NSMase2 is a key regulator that catalyzes ceramide formation, a critical step in extracellular vesicle biogenesis and release [21,26]. To determine whether ELSM treatment increases nSMase2 expression in C-MSC, we measured the time course of changes in nSMase2 protein levels after ELSM treatment. Western blotting analysis revealed that the expression of nSMase2 was similar in C-MSC ELSM and C-MSC Ctrl over the first 24 h. However, at 48 h, expression levels began to diverge, and at 72 h, there was a significant increase in nSMase2 protein levels in C-MSC ELSM compared with C-MSC Ctrl ( Figure 3A,B).

ELSM Increases nSMase2 Expression in C-MSC
NSMase2 is a key regulator that catalyzes ceramide formation, a critical step in extracellular vesicle biogenesis and release [21,26]. To determine whether ELSM treatment increases nSMase2 expression in C-MSC, we measured the time course of changes in nS-Mase2 protein levels after ELSM treatment. Western blotting analysis revealed that the expression of nSMase2 was similar in C-MSC ELSM and C-MSC Ctrl over the first 24 h. However, at 48 h, expression levels began to diverge, and at 72 h, there was a significant increase in nSMase2 protein levels in C-MSC ELSM compared with C-MSC Ctrl ( Figure 3A

ELSM-Induced EV Secretion Is Dependent on nSMase2
To determine whether increased EV secretion in C-MSC ELSM was mediated by increased expression of nSMase2 in C-MSC ELSM , we knocked down nSMase2 expression in C-MSCs with siRNAs targeting nSMase2. We measured nSMase2 mRNA levels in C-MSC by qRT-PCR, which confirmed that nSMase2 mRNA was decreased by about 70% in si-nSMase2-treated C-MSC Ctrl compared to si-NT-treated C-MSC Ctrl . Additionally, there was ~80% downregulation of nSMase2 mRNA in si-nSMase2-treated C-MSC ELSM compared to si-NT-treated C-MSC ELSM , indicating the efficiency of si-nSMase2 in knocking down nSMase2 in C-MSC treated with or without ELSM ( Figure 4A).
To determine whether ELSM-stimulated EV release is nSMase2-dependent, we quantified acetylcholine esterase (AChE) activity in EV in CM from C-MSC [27]. In control cells, ELSM increased AChE activity in EV, consistent with a stimulatory effect on EV secretion ( Figure 4B). Interestingly, nSMase2 knockdown completely abolished the ability of ELSM to stimulate EV secretion, indicating that the ELSM-induced increase in EV release from C-MSCs is dependent on nSMase2 ( Figure 4B).

ELSM-Induced EV Secretion Is Dependent on nSMase2
To determine whether increased EV secretion in C-MSC ELSM was mediated by increased expression of nSMase2 in C-MSC ELSM , we knocked down nSMase2 expression in C-MSCs with siRNAs targeting nSMase2. We measured nSMase2 mRNA levels in C-MSC by qRT-PCR, which confirmed that nSMase2 mRNA was decreased by about 70% in si-nSMase2-treated C-MSC Ctrl compared to si-NT-treated C-MSC Ctrl . Additionally, there was 80% downregulation of nSMase2 mRNA in si-nSMase2-treated C-MSC ELSM compared to si-NT-treated C-MSC ELSM , indicating the efficiency of si-nSMase2 in knocking down nSMase2 in C-MSC treated with or without ELSM ( Figure 4A).
To determine whether ELSM-stimulated EV release is nSMase2-dependent, we quantified acetylcholine esterase (AChE) activity in EV in CM from C-MSC [27]. In control cells, ELSM increased AChE activity in EV, consistent with a stimulatory effect on EV secretion ( Figure 4B). Interestingly, nSMase2 knockdown completely abolished the ability of ELSM to stimulate EV secretion, indicating that the ELSM-induced increase in EV release from C-MSCs is dependent on nSMase2 ( Figure 4B).

nSMase2 Is Directly Involved in the Modulation of Apoptosis in HL-1 Cells by the CM from ELSM-Treated C-MSC
We observed that CM from C-MSC ELSM inhibited hypoxia-induced apoptosis in cardiomyocytes in a manner that was dependent on EV secretion. To investigate whether increased SMase2 expression is responsible for the protective effects of CM from C-MSC ELSM against apoptosis in cardiomyocytes, we compared hypoxia-induced apoptosis in HL-1 cardiomyocytes treated with CM from C-MSC Ctrl si-NT , C-MSC Ctrl si-nSMase2 , C-MSC ELSM si-NT , and C-MSC ELSM si-nSMase2 . Importantly, there was no significant difference in apoptosis between the C-MSC Ctrl si-nSMase2 and C-MSC ELSM si-nSMase2 groups, suggesting that nSMase2 is directly involved in the modulation of apoptosis in HL-1 cells by the CM from ELSM-treated C-MSC ( Figure 5A-E).

nSMase2 Is Directly Involved in the Modulation of Apoptosis in HL-1 Cells by the CM from ELSM-Treated C-MSC
We observed that CM from C-MSC ELSM inhibited hypoxia-induced apoptosis in cardiomyocytes in a manner that was dependent on EV secretion. To investigate whether increased SMase2 expression is responsible for the protective effects of CM from C-MSC ELSM against apoptosis in cardiomyocytes, we compared hypoxia-induced apoptosis in HL-1 cardiomyocytes treated with CM from C-MSC Ctrl si-NT , C-MSC Ctrl si-nSMase2 , C-MSC ELSM si-NT , and C-MSC ELSM si-nSMase2 . Importantly, there was no significant difference in apoptosis between the C-MSC Ctrl si-nSMase2 and C-MSC ELSM si-nSMase2 groups, suggesting that nS-Mase2 is directly involved in the modulation of apoptosis in HL-1 cells by the CM from ELSM-treated C-MSC ( Figure 5A-E).

Discussion
Electrical stimulation associated with CRT has been shown to be an effective treatment for patients with congestive heart failure (CHF; LVEF ≤ 35%), in part by producing favorable effects on cardiac metabolism. However, most patients with heart failure are not candidates for CRT, and for some of these patients, cardiac contractility modulation (CCM) is another therapeutic option involving ELSM. Indeed, a recent randomized controlled trial demonstrated that CCM improved exercise tolerance and quality of life in specific groups of heart failure patients, resulting in fewer heart failure hospitalizations [28]. Our findings indicate that ELSM increases EV secretion from C-MSC, thereby enhancing the protective effects of C-MSC-derived CM against hypoxia-induced apoptosis in cardiomyocytes. The ELSM-mediated secretion and subsequent anti-apoptotic effects of EV were dependent on nSMase2. These findings may have important implications for understanding the beneficial effects of CRT ( Figure 6).

Discussion
Electrical stimulation associated with CRT has been shown to be an effective treatment for patients with congestive heart failure (CHF; LVEF ≤ 35%), in part by producing favorable effects on cardiac metabolism. However, most patients with heart failure are not candidates for CRT, and for some of these patients, cardiac contractility modulation (CCM) is another therapeutic option involving ELSM. Indeed, a recent randomized controlled trial demonstrated that CCM improved exercise tolerance and quality of life in specific groups of heart failure patients, resulting in fewer heart failure hospitalizations [28]. Our findings indicate that ELSM increases EV secretion from C-MSC, thereby enhancing the protective effects of C-MSC-derived CM against hypoxia-induced apoptosis in cardiomyocytes. The ELSM-mediated secretion and subsequent anti-apoptotic effects of EV were dependent on nSMase2. These findings may have important implications for understanding the beneficial effects of CRT ( Figure 6). ELSM has been found to enhance cell activities, including proliferation, growth, migration, and differentiation, showing an important potential to manipulate cellular activity under normal and pathological conditions [29]. In addition, ELSM has been developed as a biophysical environmental cue for organized tissue engineering strategies with various electric field stimulation systems, which have been shown to affect the morphology, orientation, migration, and phenotype of several different cell types [30]. A recent study demonstrated that electric stimulation increased the expression of Ca 2+ handling proteins and promoted the maturation of structural, mechanical, and firing properties of humaninduced pluripotent stem cell-derived cardiac tissue [31]. Cells 2023, 12, x FOR PEER REVIEW 10 of 16 Figure 6. Schematic overview of proposed the mechanisms of ELSM-induced cardiomyocyte protection. ELSM increases expression of nSMase2, leading to increased EV secretion, which protects cardiomyocytes from hypoxia-induced apoptosis.
ELSM has been found to enhance cell activities, including proliferation, growth, migration, and differentiation, showing an important potential to manipulate cellular activity under normal and pathological conditions [29]. In addition, ELSM has been developed as a biophysical environmental cue for organized tissue engineering strategies with various electric field stimulation systems, which have been shown to affect the morphology, orientation, migration, and phenotype of several different cell types [30]. A recent study demonstrated that electric stimulation increased the expression of Ca 2+ handling proteins and promoted the maturation of structural, mechanical, and firing properties of humaninduced pluripotent stem cell-derived cardiac tissue [31].
EVs play important roles in cell-to-cell communication in tissue homeostasis and disease conditions [32]. A recent study reported that ELSM enhances neuronal cell activity mediated by EVs derived from Schwann cells [33], but how EV-based C-MSC-cardiomyocyte interactions are modulated by ELSM is poorly understood. C-MSCs reside in the cardiac niche and play a supportive role in the regulation of physiological cardiac function. They also play a crucial role in promoting cardiac repair and regeneration by differentiation and/or paracrine actions to stimulate cardiomyocyte proliferation, activation of resident cardiac stem cells, and induction of cardiac angiogenesis in pathological states, such as myocardial infarction [34]. We used a co-culture model to assess the effects of C-MSCs on HL-1 cardiomyocytes. There are two methods for co-culturing C-MSCs with cardiomyocytes: (1) direct co-culture of C-MSCs (or CM secreted from C-MSCs) with cardiomyocytes at a specific ratio, such as 1:1; and (2) indirect co-culture, where C-MSCs and cardiomyocytes are cultured in the same well but separated by a membrane that prevents cells from passing through [35]. With the first method, it is challenging to differentiate the effects of direct cell-cell contact from indirect paracrine effects. In the second method, the distance between the cells can impact the travel of vesicles that protect cardiomyocytes. In our study, we directly applied the CM from C-MSCs stimulated with ELSM or control EVs play important roles in cell-to-cell communication in tissue homeostasis and disease conditions [32]. A recent study reported that ELSM enhances neuronal cell activity mediated by EVs derived from Schwann cells [33], but how EV-based C-MSC-cardiomyocyte interactions are modulated by ELSM is poorly understood. C-MSCs reside in the cardiac niche and play a supportive role in the regulation of physiological cardiac function. They also play a crucial role in promoting cardiac repair and regeneration by differentiation and/or paracrine actions to stimulate cardiomyocyte proliferation, activation of resident cardiac stem cells, and induction of cardiac angiogenesis in pathological states, such as myocardial infarction [34]. We used a co-culture model to assess the effects of C-MSCs on HL-1 cardiomyocytes. There are two methods for co-culturing C-MSCs with cardiomyocytes: (1) direct co-culture of C-MSCs (or CM secreted from C-MSCs) with cardiomyocytes at a specific ratio, such as 1:1; and (2) indirect co-culture, where C-MSCs and cardiomyocytes are cultured in the same well but separated by a membrane that prevents cells from passing through [35]. With the first method, it is challenging to differentiate the effects of direct cellcell contact from indirect paracrine effects. In the second method, the distance between the cells can impact the travel of vesicles that protect cardiomyocytes. In our study, we directly applied the CM from C-MSCs stimulated with ELSM or control C-MSCs to cardiomyocytes. This approach did not allow us to estimate the distance that the vesicles might travel, either in vitro or in vivo, to elicit paracrine effects.
Our findings indicate that ELSM increases EV secretion from C-MSC. Enhanced EV secretion is an adaptive cellular response to stress, such as hypoxia and inflammation. Hypoxia exposure has been reported to promote EV secretion from a variety of cells, including endothelial cells, cardiac progenitor cells [36], cardiomyocytes [37], and renal proximal tubule cells [38]. Zhang M. et al. [39] reported that CD63, the biomarker of EVs, was highly expressed in the intima of pulmonary arteries of hypoxic mice in a timedependent manner compared to the normoxic mice, consistent with hypoxia-induced EV secretion. Interestingly, Panigrahi GK et al. [40] reported that hypoxia-induced EV secretion is more prominent in prostate cancer cells isolated from African Americans; moreover, lactate content was higher in EVs secreted under hypoxia, suggesting that prostate cancer cells secreted more EVs as a survival mechanism to remove metabolic waste. Tumor necrosis factor-α (TNF-α) [41], an inflammatory mediator, was reported to increase EV secretion from retinal pigment epithelial cells [42]. Moreover, oxidative stress can also regulate EV secretion. Wang R et al. [43] reported that 1 µM H 2 O 2 significantly induced the migration of normal lens epithelial cells (LECs) by EV secretion. In this study, we found that ELSM-induced EV release from C-MSC. We did not perform studies testing the effects of ELSM on the heart in vivo. However, Klein et al. [44] found that low-frequency ELSM applied to the gastrocnemius muscle increased circulating EV abundance and altered EV-associated microRNA expression.
Local and transient ELSM was reported to promote the secretion of EVs carrying transcribed mRNA and targeting peptides. To determine whether increased EV secretion protects cardiomyocytes from hypoxia-induced apoptosis, we treated cardiomyocytes under hypoxic stress with CM from C-MSC ELSM , which confirmed the beneficial paracrine effects of C-MSC ELSM , consistent with prior studies in other cell types [19]. To further determine whether EVs were responsible for the cytoprotective effects of CM from ELSMtreated C-MSC, we removed EVs from CM by ultracentrifugation and found that the anti-apoptotic effect of C-MSC ELSM -CM was significantly diminished, indicating that the enhanced cardioprotective effects of C-MSC ELSM were indeed EV dependent.
NSMase2 and ceramide are key regulators of biogenesis and the shedding of EVs in response to external stress [20]. The budding of EVs has been shown to depend on the conversion of sphingolipids to ceramides by nSMase2 [45], and blocking nSMase2 activity has been reported to reduce EV synthesis and secretion [26,[46][47][48][49]. Recent studies have shown that the hypoxia-induced increase in EV secretion is dependent on hypoxia-inducible factor-1 (HIF-1) [38]. The capacity of MSC-derived EVs to improve cardiac function is enhanced by nSMase2-dependent transfer of microRNA-210 under hypoxic treatment [26]. Interestingly, nSMase2 was also reported to regulate the entry of miR-10b into EVs derived from metastatic breast cancer cells [50,51]. However, the underlying molecular mechanisms by which ELSM enhances the release of EVs from C-MSC remain unknown. We measured nSMase2 protein levels in response to ELSM in an immunoblotting time course study, which showed that nSMase2 levels increased over 48-72 h compared with C-MSC Ctrl . We also found that EV secretion in ELSM-enhanced C-MSC was abolished by nSMase2 knockdown, suggesting a key role for nSMase2 in ELSM-induced EV secretion. Importantly, knockdown of nSMase2 abolished the protective effects of CM from ELSM-treated C-MSC, suggesting that nSMase2 is indirectly involved in the modulation of apoptosis in HL-1 cells by regulating EV secretion from ELSM-treated C-MSCs.
ELSM can also change the energy metabolism of cells. For example, ELSM was reported to alter fatty acid metabolism in isolated skeletal muscle, along with exogenous free fatty acid (FFA) oxidation, exogenous FFA incorporation into intracellular triacylglycerol (TG), and intracellular TG content in the isolated muscle fibers [52]. ELSM was also reported to increase glucose metabolism in neurons detected by micro-PET imaging of FDG uptake [53]. Global analysis of the transcriptome and proteome demonstrated that ELSM significantly regulated proteins and genes enriched in biological processes related to glycolytic pathways, fatty acid oxidation, oxidative phosphorylation, as well as autophagy/mitophagy and oxidative stress, in human myotubes [54].
Cellular stresses, such as hypoxia and inflammation, have been reported to alter the content of EVs [55]. ELSM treatment has the potential to affect DNAs, RNAs (e.g., long noncoding RNAs, mRNAs, microRNAs, etc.), lipids, and proteins (e.g., enzymes, receptors) contained in EVs from C-MSC. Previous studies have shown that ELSM altered cellular transcriptional regulation, inducing transcription factors such as NFAT3, GATA4, NRF-1 (nuclear respiratory factor 1), c-Jun, and cytochrome C, leading to increased growth and maturation of cardiomyocytes, mitochondrial proliferation, and progenitor cell differentiation [56][57][58]. Yu B et al. [59] found that EVs from GATA4-overexpressing MSC carry high concentrations of microRNAs (e.g., miR-19a, miR-451, and miR-221) and growth factors (e.g., IGF-1). Hao C. et al. [60] also demonstrated that overexpression of GATA4 enhanced the protective effect of cardiac colony-forming unit fibroblast-derived EVs against myocardial ischemic injury by transferring miR221, which inhibited PTEN expression by activating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. NRF-1 is a transcription factor that regulates the expression of mitochondrial transcription factor A (TFAM), a transcription factor required to initiate the transcription and duplication of mtDNA and thus mitochondrial synthesis [61]. A recent report showed that the expression of NRF-1 and TFAM was significantly decreased after hepatic ischemia-reperfusion injury subsequent to hepatectomy, but EVs derived from adipose-derived MSC had the ability to enhance mitochondrial biogenesis by increasing NRF-1 and TFAM expression [62]. ELSM treatment was also reported to increase the mRNA level of c-FOS/ERK1/2 in electrically stimulated cells [63], c-FOS is a transcription factor for ERK1/2. Epigenetic regulation might also be involved in ELSM-induced gene regulation. Klymenk O. et al. [64] reported that ELSM-induced interleukin 6 (IL6) transcription was enhanced in histone deacetylase 5(HDAC5)-deficient C2C12 myotubes. Recent studies have shown that EV-derived microRNAs contribute to cardioprotection by acting as anti-apoptotic mediators [65,66]. Electrical stimulation acupuncture has also been reported to increase renal blood flow via exosome-delivered miR-181 [44].
According to a recent study, inhibiting the secretion of EVs using the nSMase 2 inhibitor GW4869 resulted in a significant increase in cell senescence. This was demonstrated by showing an increase in the expression of several senescence-associated markers, including γH2AX, senescence-associated β-galactosidase (SA-β-gal), IL6, MMP13, and P16. Additionally, treating mice with GW4829 systemically for two months led to accelerated aging, as evidenced by increased staining of γH2AX and SA-β-gal in the liver, spleen, lung, and kidney. Furthermore, inhibiting EV release through knocking out the small GTPase Rab27a also accelerated senescence in various cells and mice [67]. Mitochondrial function, particularly electron transport chain (ETC) activity, is known to be important in inducing premature senescence [68]. Inhibition of EV secretion through knockdown of the small GTPase Rab27b was found to impair oxidative phosphorylation in cardiac mesenchymal stem cells and to down-regulate the expression of key genes involved in fatty acid β-oxidation, TCA, and ETC [1]. This suggests that EV secretion may have a beneficial effect against senescence by regulating metabolism.

Limitations and Future Direction
We presume that the protective effects of the conditioned medium are derived from both the quantity and quality of EVs. While it is straightforward to quantify EVs present in the conditioned medium, identifying the specific components (proteins, lipids, or RNAs) responsible for these beneficial effects poses a significant challenge. Future studies (i.e., mass spectrometry, lipidomic profiling) are required to explore the molecular content of EVs from ELSM-treated C-MSC. This will enable identification of specific mechanism of apoptosis protection by ELSM (i.e., reduction in ceramide levels).
In this study, we used a previously developed ELSM protocol [18], and future studies are needed to investigate the combination of pacing dose and pacing duration to obtain optimal pacing output, which may provide useful information for the benefit of multielectrode pacing across the left ventricle.
Meraviglia V et al. [69] reported that ELSM significantly reduced the expression of connexin43 (Cx43) by increasing the acetylation of Cx43 on HL-1 cardiomyocytes at 0.5 Hz for 24 h, suggesting that ELSM may reduce the expression of Cx43 by affecting the protein level of Cx43 and reducing cardiomyocyte-to-cardiomyocyte communication. Another report suggested that Cx43 expression was highly sensitive to ELSM in a frequencydependent manner that high stimulation frequency leads to a substantial reduction in global Cx43 expression in murine-induced pluripotent stem cell-derived cardiomyocytes by