Mechanistic convergence of exercise and mesenchymal stem cell-derived exosome signaling in isoproterenol-induced myocardial injury

Abstract

Ischemic heart disease remains the most significant cause of morbidity and mortality worldwide. Although conventional therapies such as β-blockers, angiotensin inhibitors, statins, and percutaneous coronary intervention have reduced mortality in industrialized nations, progress has plateaued, and global ischemic burden continues to rise. Recent advances in molecular biology have enabled mechanistic interrogation of canonical cardioprotective signaling pathways at a resolution not previously achievable. Parallel advances in exercise biology and mesenchymal stem cell-derived exosomes (MSC-EXO) research suggest an opportunity for synergistic cardioprotection. Exercise operates as a complex molecular stimulus that can activate redox-sensitive kinases, autophagy regulators, and metabolic stimulus that extends beyond its classic descriptive cell-autonomous kinase activation and metabolic remodeling within the cardiomyocyte itself. Exercise is now recognized as a multi-organ secretome-generating stimulus that mobilizes circulating extracellular vesicles, exerkines, and microRNA-loaded exosomes as systemic paracrine mediators capable of signaling across tissue boundaries. This reframing establishes exercise biology and MSC-EXO research as fields operating through a shared biological currency called vesicle-mediated intercellular communication. State-of-the-art investigations have sought to decode this “exercise secretome” and develop tools to modulate these chemical cascades. Multiple experimental studies report that MSC-EXO function as biologically active paracrine vectors that deliver regulatory microRNAs and proteins to recipient cells and promote angiogenesis, suppress apoptosis, and support mitochondrial function. This mechanistic review assesses the effectiveness of interventions on canonical pathways such as ERK and AKT/mTOR on isoproterenol (ISO)-induced myocardial ischemia models to the heart based on recent animal and human studies. Supporting literature on stem cell biology, exosome delivery strategies, and translational barriers is discussed to construct an integrated mechanistic framework that demonstrates how this therapy reinforces and sustains reparative signaling in an ISO-induced ischemic environment. Exercise biology and MSC-EXO research represent a synergistic therapeutic strategy with the potential to advance regenerative cardioprotection in ischemic cardiomyopathy, wherein, exercise-induced kinase sensitization and metabolic preconditioning establish a permissive intracellular environment, and targeted exosome delivery amplifies the same ERK, PI3K-Akt, and mTOR survival networks to sustain reparative signaling beyond what either intervention achieves independently.

Key Words: Myocardial ischemia; Isoproterenol; Mesenchymal stem cell; Exosomes; Nanoparticles; ERK; Exercise; AKT/mTOR

Core Tip: Exercise primes cardiomyocyte stress tolerance through ERK and PI3K/Akt/mTOR pathway activation, regulated autophagy, and mitochondrial preservation, while mesenchymal stem cell-derived exosomes reinforce these same survival networks via paracrine delivery of bioactive cargo. Integrating exercise conditioning with targeted, cell-free exosome-based strategies may overcome current therapeutic plateaus by enhancing biological engagement, improving myocardial resilience, and biasing post-ischemic remodeling toward adaptive recovery.

INTRODUCTION

Ischemic heart disease (IHD) remains a leading cause of global morbidity and mortality and continues to impose a substantial clinical and economic burden despite advances in medical and interventional therapy[1-3]. According to the World Health Organization (WHO), cardiovascular diseases accounted for approximately 19.8 million deaths worldwide in 2022, representing 32% of all global deaths, with 85% attributable to IHD and stroke[4]. While established treatments such as β-blockers, renin-angiotensin system inhibitors, statins, and contemporary revascularization strategies have improved survival, substantial residual risk persists, and long-term functional recovery remains incomplete for many patients[1,2,5]. The WHO reports that cardiovascular disease accounts for at least 38% of premature deaths (< 70 years) due to noncommunicable diseases[4]. This persistent global burden has shifted investigative focus toward regenerative and biologically targeted strategies aimed at augmenting endogenous myocardial repair. Strategies focused primarily on acute ischemic injury, such as revascularization, thrombolysis, and early infarct limitation, do not adequately address downstream processes including mitochondrial dysfunction, oxidative stress, maladaptive remodeling, impaired angiogenesis, dysregulated autophagy, and loss of cardiomyocyte functional reserve[1,6,7].

Contemporary investigation has moved toward mechanistic exploitation of the shared paracrine and exosome-mediated signaling, such as the ERK and PI3K-Akt-mTOR signaling networks in the intracellular survival and repair pathways that govern cardiomyocyte fate following ischemic injury[2,8,9]. In parallel, non-pharmacologic and regenerative approaches such as structured physical activity and mesenchymal stem cell (MSC) based therapies have demonstrated promising avenues to address the therapeutic plateau observed with conventional interventions[1,6,7]. The field has shifted beyond descriptive assessments of exercise and cell therapy and instead emphasizes exploiting the mechanistic convergence of paracrine and exosome-mediated signaling in integrative cardioprotection[2,6]. In this framework, MSC-derived exosomes (MSC-EXO) are recognized as principal mediators of therapeutic effect, delivering defined molecular cargo that activates pro-survival and reparative signaling pathways and whose efficacy can be enhanced through targeted engineering and delivery strategies[10].

Isoproterenol (ISO)-induced myocardial ischemia is a widely used experimental model that reproduces key features of ischemic cardiac injury, including β-adrenergic overstimulation, oxidative stress, mitochondrial dysfunction, and cardiomyocyte necrosis[2,8]. Within this context, endogenous survival pathways are activated in response to injury but are often insufficient to prevent adverse remodeling and functional decline, rendering the model a vigorous platform for evaluating targeted cardioprotective interventions[8,11].

Taken together, these observations support a unifying scaffolding in which cardioprotection in ISO-induced myocardial injury can be understood through the lens of convergent intracellular survival signaling. In this review, we first contextualize ERK and PI3K-Akt-mTOR pathway activation within the pathophysiology of β-adrenergic mediated myocardial stress. We then examine how exercise conditioning reshapes these kinase networks to enhance metabolic resilience and regulated autophagy, and how MSC-EXO further reinforce these same pathways through targeted paracrine signaling. We then propose a model of synergistic cardioprotection that integrates these domains in which exercise establishes a permissive intracellular environment that may amplify the reparative signaling delivered by MSC-EXO, with consideration of translational strategies that could optimize biological engagement in ischemic myocardium.

LITERATURE REVIEW

This study was designed as a focused narrative review, given the heterogeneity of experimental models, variability in exercise paradigms, and evolving understanding of paracrine and exosome-mediated mechanisms underlying cardioprotection in IHD. The review aimed to synthesize mechanistic and translational evidence examining the convergence of exercise-induced signaling and MSC-EXO pathways in the context of ISO-induced myocardial ischemia, with particular emphasis on ERK and Akt-mTOR signaling networks.

Peer-reviewed original research articles, randomized and non-randomized clinical studies, animal models, experimental mechanistic studies, and narrative or translational reviews were eligible for inclusion if they addressed at least one of the following domains: Exercise-induced cardioprotective signaling, MSC or MSC-EXO-mediated paracrine effects, ISO-induced myocardial injury, or ERK-Akt-mTOR pathway modulation in ischemic contexts. Studies focusing exclusively on non-cardiac systems, unrelated disease models, or lacking mechanistic relevance were excluded.

Article screening and selection were performed by the authors based on relevance to the study objective. When questions regarding eligibility arose, inclusion was determined by consensus discussion. Given the narrative and mechanistic nature of the review, no formal risk-of-bias assessment or quantitative meta-analysis was undertaken.

The included literature was synthesized thematically to contextualize current understanding of cardioprotection according to metabolic adaptation, paracrine and exosome-mediated signaling, intracellular survival pathways, and structural remodeling outcomes following ischemic stress. Key interventions identified across the included literature are summarized in Table 1.

Table 1 Summary of interventions.

Intervention	Mechanism/procedure	Benefit	Drawback	Ref.
Intramyocardial injection	Direct injection of stem cells into damaged myocardium	Direct targeted infusion of a large number of cells	Mechanical tissue damage; difficult infarct localization; higher arrhythmia incidence; suboptimal electromechanical coupling post-injection	[7]
Transendocardial intramyocardial injection	Percutaneous catheter-based, image-guided injections into endocardial surface targeting borderzone myocardium	Precise border zone access without open chest surgery; reported improvements in perfusion, EF, diastolic/LV function, and 6-minute walk metrics in preliminary work	Preliminary stage with inconsistent results	[7]
Epicardial intramyocardial injection	Surgical direct-visual injection into infarcted myocardium, typically adjunct during open-heart surgery	Direct visual access; described as not having coronary embolism risk	More invasive; leakage; dosing control challenges; inadequate donor-cell retention; studies limited to animals	[7]
Intrapericardial injection	Intrapericardial delivery of hydrogel compound containing MSCs; compared vs intramyocardial control; porcine feasibility/safety reported	Approximately 10 × higher viable cell retention vs intramyocardial in mice; increased exosome secretion and reduced myocardial apoptosis; no major adverse effects in mice; no abnormal cardiac events in pigs over 4 days	Follow-up in pigs only 4 days, so long-term effects not assessed	[21]
Intracoronary infusion	Catheter-based delivery into infarct-related artery by inflating the catheter and infusing cells during inflation; used during catheterization	Human trials described with EF increases, reduced scarred myocardium, and lack of arrhythmia post-procedure	Long-term effects not established; procedural blinding was limited; > 65 excluded; concern raised about potential bias in reporting/editorial handling; animal data suggest possible microvascular obstruction and myocardial injury	[12,28]
Embryoid body formation	Generate iPSC aggregates to drive spontaneous lineage commitment, then isolate MSC-like cells	Simple; cost-effective	Heterogeneity; scale-up challenges; limited microenvironment control	[19,24]
Specific differentiation	Predifferentiate iPSCs toward a lineage, then apply factors/conditions to yield iMSCs	Greater regenerative potential	More time-consuming; higher cost	[19,25]
Blood-based method	Culture iPSCs under blood-derived supplement conditions to promote iMSC phenotype	Low-cost; high proliferative potential	Potential immune reactivity if residual blood components/cell fragments persist	[25]
MSC switch method	Switch iPSC medium to MSC growth media; optional FACS to select subpopulations	Operationally straightforward; selection can improve consistency	Variability in signaling potency and paracrine profile; regulatory classification changes	[19,25]
Pathway inhibitor method	Use chemical pathway inhibitors to drive iPSC to iMSC differentiation	Can reduce heterogeneity via controlled signaling	Labor-intensive; scale-up/yield limitations	[19,25]
UTMD microbubble gene delivery	IV gene-loaded cationic microbubbles; ultrasound cavitation destroys bubbles to deliver genes	Enhances MSC homing; improved repair outcomes vs single-gene	Theoretical toxicity/embolism; transient gene expression	[8]
Targeted nanobubble-exosome delivery	IV nanobubble-antibody-exosome complex; LIPUS disrupts nanobubbles to drive exosome release/penetration	Improves myocardial retention/uptake vs controls; reduces non-cardiac sequestration	Preclinical window and assays limit safety claims; off-target biodistribution remains plausible	[10]
Dual-membrane phase-change nanoparticles	IV phase-change nanoparticles with MSC + macrophage membranes; miRNA-125b surface-adsorbed	Anti-apoptotic and anti-fibrotic effect	Short miRNA activity; repeat dosing	[14]

MSC: Mesenchymal stem cell; iPSC: Induced pluripotent stem cell; iMSC: Induced mesenchymal stem cell; EF: Ejection fraction; LV: Left ventricular; FACS: Fluorescence-activated cell sorting.

MOLECULAR PATHOPHYSIOLOGY OF ISO-INDUCED MYOCARDIAL INJURY AND SURVIVAL PATHWAYS

ISO is a synthetic β-adrenergic agonist that induces myocardial injury through sustained catecholaminergic stimulation, resulting in calcium overload, excessive reactive oxygen species (ROS) production, mitochondrial impairment, and cardiomyocyte death[2,8]. These mechanisms recapitulate key neurohumoral and oxidative components of human IHD, and support the translational relevance of the ISO model for mechanistic studies[2,11].

In ISO-induced injury, cardiomyocytes experience increased metabolic demand, impaired adenosine triphosphate (ATP) generation, mitochondrial dysfunction, and activation of stress-responsive signaling cascades[8,9]. In response, intracellular survival pathways, including ERK and PI3K-Akt-mTOR, are engaged in an attempt to restore cellular homeostasis and limit injury progression[2,8,9]. Autophagy represents a central component of this adaptive response, as regulated removal of damaged proteins and organelles is essential for maintaining cardiomyocyte viability under ischemic stress[2,9]. However, dysregulation of autophagy can exacerbate injury, highlighting the importance of precise temporal and quantitative control of these pathways[9].

EXERCISE-INDUCED MODULATION OF TWO INTRACELLULAR SURVIVAL PATHWAYS: THE ERK PATHWAY, AND THE PI3K, AKT, AND mTOR SIGNALING PATHWAY IN ISO-INDUCED MYOCARDIAL INJURY

Exercise promotes physiological cardiac remodeling and enhances resistance to ischemic stress through coordinated activation of intracellular survival pathways that overlap with those engaged during ischemic injury[1,6,7]. In the context of ISO-induced myocardial stress, exercise conditioning modulates redox-sensitive and energy-responsive kinases, including ERK, PI3K-Akt, AMPK, and mTOR, thereby improving cardiomyocyte stress tolerance and metabolic flexibility[2,9].

Exercise-induced activation of ERK and PI3K-Akt support cytoprotection through enhanced mitochondrial efficiency, improved ATP generation, and stabilization of calcium handling under catecholaminergic stress[2,7,9]. Concurrent regulation of mTOR signaling and AMPK activity promotes balanced autophagic flux, which is critical for removal of damaged proteins and mitochondria during ischemic stress[2,9]. Rather than preventing ISO-mediated injury outright, exercise conditioning shifts cardiomyocytes toward a phenotype characterized by enhanced signaling plasticity, improved mitochondrial quality control, and greater resistance to oxidative and apoptotic triggers[1,6,7].

These signaling adaptations establish a biologically permissive intracellular environment in which pro-survival pathways are more rapidly and effectively engaged during subsequent ischemic challenge, providing a mechanistic basis for exercise-mediated cardioprotection in ISO models[2,9].

MSC-EXO AS PARACRINE REINFORCERS OF ERK AND PI3K, AKT, AND mTOR SURVIVAL NETWORKS

MSC-based therapies represent an area in regenerative cardiovascular medicine that demonstrates therapeutic benefit predominantly through paracrine mechanisms rather than durable cardiomyocyte engraftment[1,2,6,12,13]. Among these mediators, MSC-EXO function as biologically active vesicles capable of delivering regulatory microRNAs, heat shock proteins, antioxidative enzymes, and survival-associated adaptor proteins to injured myocardium[6,10,14].

In ischemic settings, MSC-EXO enhance angiogenesis, attenuate apoptosis and fibrosis, and preserve mitochondrial function, effects that converge mechanistically on ERK and PI3K-Akt-mTOR signaling cascades[2,9,10,14]. Exosomal cargo has been shown to modulate PTEN activity, regulate PIP2-PIP3 balance, reinforce Akt phosphorylation, and support downstream transcriptional programs associated with mitochondrial biogenesis and anti-apoptotic signaling[2,9,10]. Parallel activation of ERK signaling further promotes cytoprotective remodeling and stress-response adaptation[2,9].

These paracrine inputs amplify endogenous survival pathways that are insufficiently activated during sustained β-adrenergic overstimulation, thereby reinforcing intracellular repair signaling in ISO-induced myocardial injury[2,8,9]. Advances in delivery engineering, including ultrasound-targeted and nanoparticle-based platforms, aim to enhance myocardial retention and signaling potency of these vesicles in translational models[10,14].

TRANSLATIONAL OPTIMIZATION OF EXOSOME SIGNALING IN CARDIAC ISCHEMIA

Stem cell source considerations for exosome-based therapy in cardiac ischemia

Pluripotent vs multipotent stem cells: Pluripotent stem cells (PSC) can generate tissue from all three germ layers and differentiate into nearly any somatic cell type, whereas multipotent stem cells are restricted to lineage-specific differentiation[6]. Among multipotent populations, MSCs, specifically bone marrow-derived and adipose-derived MSCs (AD-MSCs), are the most extensively studied in cardiac and connective tissue research, owing to their relative ease of isolation and expansion, low immunogenicity permitting allogeneic use, favorable safety profile with low tumorigenic risk, and potent paracrine signaling that supports angiogenesis, immunomodulation, and tissue repair (Table 1)[2].

Advantages and limitations of adipose vs bone marrow-derived MSCs: MSCs are adult stem cells capable of differentiating into mesodermal lineages, such as bone, cartilage, muscle and adipose. However, their cardioprotective effects are not primarily mediated by direct cardiomyocyte differentiation but are instead indirectly mediated through paracrine signaling. MSCs secrete exosomes and other soluble factors that support cardiomyocyte survival, promote angiogenesis, recruit endogenous progenitor cells, and reduce apoptosis in injured myocardium.

While bone marrow was historically the primary MSC source, AD-MSCs are an attractive alternative due to higher cell yields, less invasive harvesting, and greater proliferative capacity[6,15]. Stem cells also have differences in their morphology, surface marker expression, and differentiation potentials, whereas AD-MSCs are structurally and phenotypically similar to bone marrow-derived cells[15]. Despite these advances, senescence is still a limiting factor in treatment with AD-MSCs. The issue may be pronounced in ischemic myocardium, where excessive branched-chain amino acid is thought to accumulate, or in the loss of H3K9me3, a histone modification mark that maintains heterochromatin, that may accelerate senescence and impair MSC therapeutic efficacy[9].

Methods of delivery

Successful therapeutic signaling from MSC-based approaches, including cell-derived exosomes and other soluble factors, depends not only on the cell cargo but also on the delivery method[5]. Poor engraftment has served as a barrier to therapeutic usability due to untargeted delivery and low cell retention at injury sites. Many methods of delivery have been created to bring the administered cells to their targeted tissue. These methods of delivery come with various advantages and limitations as detailed in the following sections[16].

Intramyocardial injection approaches: Intramyocardial injection involves directly injecting stem cells into the damaged myocardium[17]. This modality is currently being tested on human subjects. The benefits of this method are the direct targeted infusions of a large amount of cells. Issues with this method can involve mechanical damage to the tissue, difficulty identifying infarcted tissue during the procedure, and challenges with electromechanical integration with the host myocardium post-injection. Higher incidences of arrhythmias have been reported in some intramyocardial injection studies depending on the cell product used. Two main routes are currently used: The transendocardial and the epicardial injection[7,18].

The transendocardial route uses catheter-based, image-guided injections directly into the endocardial surface. This will enable precise access to the borderzone myocardium without requiring open chest surgery. It is performed percutaneously through peripheral vessels such as the femoral artery or vein. The catheter is guided to the infarct zone using a fluoroscopic two-dimensional or three-dimensional (3D) system. The 3D system may provide a more even distribution of cells around the zone of injury. It should be noted that the research is still in a preliminary stage, and consistent results have not yet been found. Some of the benefits already seen from this are improved perfusion, increased ejection fraction, improved diastolic function and left ventricular function, improved 6 Minute Walk scores, and improved Minnesota Living with Heart Failure Questionnaire scores[7,19,20].

The epicardial route provides direct visual access to the infarcted myocardium during surgery. It involves using a 27 gauge-bent needle to inject into the infarcted myocardium. This method is more invasive, and is typically done during a left thoracotomy or sternotomy as an adjunct treatment during an open heart surgery. Risks for this treatment include leakage, difficulty controlling precise dosages, and the inadequate retention of donor cells. This approach avoids direct intracoronary infusion related embolization risk. Studies are predominantly preclinical[7].

Intrapericardial catheter-based delivery: The intrapericardial (IPC) route offers a minimally invasive approach that maximizes stem cell retention while limiting myocardial damage[18]. In a murine myocardial infarction model, IPC delivery of MSCs in a hydrogel compound achieved approximately 10-fold higher viable cell retention compared with intramyocardial injection, along with increased exosome secretion and reduced myocardial apoptosis. A subsequent porcine feasibility study demonstrated no adverse cardiac events during a 4-day monitoring period, though long-term effects and follow-up was not established in the study[21].

Intracoronary: The intracoronary route delivers stem cells into the infarct-related coronary artery during catheterization, typically as a divided infusion through a coronary catheter to promote downstream distribution within the infarct territory[12]. The British Medical Journal issued an Expression of Concern and opened a content-integrity investigation citing data-integrity and disclosure or authorship concerns, and cautioned that the results may not be reliable[22]. In a randomized post-acute myocardial infarction trial using intracoronary Wharton’s jelly MSC infusion, improvements in left ventricular ejection fraction at 6 months were reported compared with conventional care. A larger improvement was observed when a booster infusion was given, and no procedure-related arrhythmias or coronary flow compromise were reported. These functional gains are commonly interpreted as being mediated primarily by paracrine signaling rather than durable cardiomyocyte engraftment[1,12]. In the TAHA8 trial, procedural blinding was limited and participants older than 65 years were excluded, which may increase risk of bias and limit generalizability[13]. Animal studies have also shown a possibility of microvascular obstruction and myocardial injury. Despite these limitations, intracoronary delivery remains clinically relevant. It can be performed during standard catheterization workflows and provides targeted delivery to the infarct-related coronary circulation compared with systemic infusion[12,13].

Intravenous: Intravenous delivery offers a minimally invasive, repeatable administration route that may promote systemic cardioprotective signaling. Preclinical studies demonstrate improved left ventricular function and reduced fibrosis following repeated IV infusions, suggesting a cumulative therapeutic benefit. However, cardiac homing is limited, with only a small fraction of administered cells reaching the target myocardium[11].

Advancements

Stem cell senescence: Though AD-MSCs are still widely used for stem cell research, current preclinical research is exploring induced MSCs (iMSCs). iMSCs are generated by reprogramming somatic cells into induced PSCs (iPSCs), then differentiating them into MSC-like cells. iMSC derivation from iPSCs can enable greater expansion capacity and mitigate donor-limited proliferation and senescence seen in primary MSC preparations. Five methods of acquiring these cells are embryoid body formation, specific differentiation, blood-based method, MSC switch method, and pathway inhibitor method. Each method has advantages and drawbacks[23].

Embryoid body formation uses 3D aggregates of PSCs (including iPSCs) that undergo spontaneous differentiation[3]. This method is simple and cost-effective. There may be difficulty obtaining homogeneity in body size and shape and scaling up embryoid body production. There is also difficulty achieving full control over the microenvironment of embryoid bodies[24,25].

The specific differentiation approach involves pre-differentiation of iPSCs into a particular lineage, then growing stem cells using growth factors to differentiate them into iMSCs[24]. Cells generated via this method are more time- and resource-intensive to produce, and some reports suggest lineage-directed differentiation may yield iMSCs with enhanced regenerative properties[23,25,26].

The blood-based method uses a culture containing blood-based supplements, such as human platelet lysate, to make iMSCs. This is another low-cost method that has high proliferative potential, but may trigger immune reactions due to possible failures in removing cell fragments such as platelets[23].

The MSC switch method induces iMSC differentiation by replacing iPSC culture medium with MSC growth media. One such medium is DMEM/MEM/IMDM supplemented with FBS, bFGF, TGF-β1, insulin, transferrin, penicillin, phenol red, and serum. This operationally straightforward approach can be combined with FACS to select specific subpopulations for improved consistency. However, variability in signaling potency, regulatory classification, and paracrine composition remains a challenge[23,26].

The pathway inhibitor technique uses chemical inhibitors of certain pathways to facilitate differentiation of iPSCs into iMSCs. Use of this approach is laborious and may present issues of obtaining large quantities of usable cells, but this method provides a mechanism to reduce heterogeneity among the cells[23,25,26].

Bubble technology: Ultrasound-targeted bubble destruction has been investigated as a noninvasive delivery modality in preclinical models[8,10]. Gene cargo was used to enhance MSC homing to cardiac tissue[8]. Through these bubble carriers, cargo such as shFOXO4 and SDF1 may be delivered to the myocardium via intravenous injection[8]. In these two preclinical studies, bubble carriers were implemented as microbubbles, which carry gene cargo, and nanobubbles, which carry exosome cargo[8,10]. Figure 1 provides a schematic overview of the three ultrasound-enabled delivery platforms discussed below, linking each carrier (microbubble, nanobubble, phase-change nanoparticle) to its therapeutic payload and the proposed downstream biologic effect.

Figure 1 Ultrasound-enabled delivery platforms for myocardial repair signaling (schematic). Schematic comparison of: (1) Targeted nanobubbles carrying adipose-derived mesenchymal stem cell exosomes enriched for SDF-1α via anti-CD81 tethering and cRGD targeting, with LIPUS used to increase myocardial localization and exosome availability; (2) Cationic microbubbles carrying SDF1 and shFOXO4 plasmids delivered by ultrasound-targeted microbubble destruction; (3-6): Enabling cellular entry (3) and transcription (4), with SDF-1α secretion supporting chemotactic homing (5) and ShFOXO4 driving FOXO4 knockdown to promote senescent-cell clearance (6); and (7) Phase-change nanoparticles delivering miR-125b to support post-transcriptional silencing and reduced intrinsic apoptosis signaling. This figure is a conceptual synthesis and is not intended to imply that every intermediate node shown was directly measured in each cited study. This figure was created by BioRender.com (Supplementary material). LIPUS: Low-intensity pulsed ultrasound; UTMD: Ultrasound-targeted microbubble destruction; SDF-1α: Stromal cell-derived factor-1α; AD-MSC: Adipose-derived mesenchymal stem cell; ROS: Reactive oxygen species; MOMP: Mitochondrial outer membrane permeabilization.

Microbubbles are lipid-membrane, gas-core cationic bubbles that can be used to carry plasmid genes. The microbubbles can be destroyed via cavitation triggered by an ultrasound. shFOXO4 knockdown plus SDF1 overexpression was used as a rejuvenation pretreatment to reduce senescence and enhance SDF1-mediated chemotactic recruitment of MSCs to the aging heart. This dual-gene strategy was associated with improved cardiac repair outcomes relative to single-gene treatment. Benefits of gene preconditioning may improve MSC homing efficiency, reduce senescence, lower inflammation, enhance angiogenesis, and reduce infarct relative to the aged heart. While not observed in studied specimens, researchers theorize a systemic toxicity from microbubbles and embolism may develop as a part of therapy. Because gene expression is transient after delivery, durability is limited, and treatment may require optimized dosing schedules for efficient therapy[8].

Nanobubbles are lipid-shelled, gas-core bubbles that are smaller than microbubbles. Nanobubbles can carry MSC-EXO to the myocardium intravenously through a nanobubble-antibody-exosome complex. The nanobubble is first made using a biotinylated lipid shell. Streptavidin is then incubated with the nanobubble to allow for additional binding and is conjugated to the biotinylated shell. Next, an anti-CD81 antibody is bound to streptavidin that serves to tether the MSC-EXO and form the targeted nanobubbles. Finally, the MSC-EXO are incubated with the bubbles to mount the exosomes, and the bubbles are now fully primed for delivery.

Following intravenous injection, low-intensity pulsed ultrasound is used to disrupt nanobubbles and enhance local exosome release and uptake. This method was designed to improve myocardial retention and uptake of exosomes. It addresses a specific issue with the intravenous delivery of exosomes, where exosomes may accumulate in non-cardiac organs. No toxicity was observed on organ histology with supportive routine blood and biochemical testing. Although, the absence of detected adverse effects should be interpreted within the scope of the assays performed and the study’s preclinical assessment window, and off-target biodistribution remains a plausible limitation for exosome-based therapeutics[10].

Nanoparticles: Phase-change nanoparticles can be engineered as MSC-membrane-coated carriers that deliver therapeutic cargo such as miRNA-125b to injured myocardium. This may augment downstream repair pathways relevant to stem cell-based cardiac regeneration. These particles consist of a perfluorocarbon phase-change core encased in a dual MSC-macrophage membrane coating, with miRNA-125b attached to the nanoparticle surface via charge adsorption. miRNA-125b, an endogenous MSC-secreted microRNA, exerts cardioprotective effects by downregulating apoptosis-related proteins and inhibiting fibroblast proliferation. Following intravenous injection, the nanoparticles deliver this therapeutic cargo to injured myocardium; however, the short active duration of miR-125b necessitates repeated dosing[14].

DISCUSSION

Integrative model for combining exercise and MSC-EXO therapy in ISO-induced myocardial ischemia

The model proposed in this study conceptualizes cardioprotection as a coordinated biological process in which exercise and MSC–EXO signaling act in concert to reinforce cardiomyocyte survival and bias post-injury remodeling toward adaptive outcomes in ISO–induced myocardial ischemia (Figure 2). Exercise and regenerative therapies are not independent interventions, but biologically synergistic processes that share intracellular kinase networks and mitochondrial regulatory nodes[1,2,8,24].

Figure 2 Integrative signaling model of exercise conditioning and mesenchymal stem cell-derived exosome therapy in isoproterenol-induced myocardial ischemia. (1) Exercise conditioning activates RTK signaling through growth factor pathways, including EGFR, FGFR1/2, and VEGFR, thereby priming downstream intracellular survival pathways; (2) RTK activation engages RAF-MEK-ERK and PI3K-Akt-mTOR signaling cascades that regulate cytoprotection, metabolic adaptation, and autophagic balance; (3a) Mesenchymal stem cell-derived exosomes (MSC-EXO) modulate cardiomyocyte signaling at the plasma membrane through RTK antagonism or receptor interference; (3b) In parallel, exosomes undergo vesicle fusion with the plasma membrane followed by endosome formation, enabling intracellular cargo delivery; (4) Exosomal cargo includes heat shock proteins, antioxidative enzymes, survival-associated adaptor proteins, and microRNAs that modulate intracellular signaling networks; (5) These paracrine inputs suppress negative regulators like PTEN to balance PIP2 and PIP3 levels, which reinforces Akt pathway activity. Meanwhile, isoproterenol-induced β₁-adrenergic overstimulation activates adenylyl cyclase and the cAMP-PKA pathway, which causes calcium influx through long-lasting (L-type) channels and leads to sustained intracellular calcium loading; (6) Excess mitochondrial Ca²⁺ impairs electron transport chain complexes II and III, which causes electron leak and reactive oxygen species generation, which drives oxidative damage, mitochondrial DNA injury, and activation of pro-apoptotic signaling pathways; (7) Exercise-primed signaling and MSC-EXO cargo counteract these processes by enhancing antioxidant defenses, stabilizing mitochondrial function, and upregulating anti-apoptotic and mitochondrial biogenesis-associated transcriptional programs, including BCL-2, BCL-xL, NRF1, and TFAM, in part through p53-dependent regulation; and (8) ERK and Akt-mTOR signaling support regulated autophagy, limit apoptosis and fibrosis, preserve mitochondrial integrity, and promote adaptive remodeling and functional recovery following myocardial ischemic injury. This figure was created by BioRender.com (Supplementary material). MSC: Mesenchymal stem cell; ISO: Isoproterenol; ROS: Reactive oxygen species.

Exercise enhances baseline responsiveness of the RAF-MEK-ERK and PI3K-Akt-mTOR pathways, which collectively regulate cytoprotection, metabolic flexibility, and autophagic homeostasis[1,2,24]. Although these adaptations do not prevent myocardial injury under conditions of sustained β-adrenergic overstimulation, they shift cardiomyocytes toward a state of increased stress tolerance and signaling plasticity. When these pathways are engaged, they promote cardiomyocyte survival, preserve mitochondrial integrity through regulated quality control mechanisms, and limit excessive extracellular matrix deposition. These effects support maintenance of ventricular structure and contractile function following ISO-induced myocardial injury[1,2,8].

Beyond direct cardiomyocyte stress signaling, ISO-induced myocardial injury is characterized by secondary inflammatory and remodeling cascades that contribute to adverse structural outcomes[6,7]. Sustained oxidative stress promotes cytokine release and chemokine-driven leukocyte recruitment, amplifying local inflammatory signaling within the myocardium. Platelet–leukocyte crosstalk, thrombin generation, and fibrin-mediated activation further contribute to microvascular dysfunction and extracellular matrix remodeling under conditions of persistent β-adrenergic overstimulation[6,7].

Dysregulated inflammatory signaling promotes fibroblast activation, excessive extracellular matrix deposition, and transition from adaptive hypertrophy toward fibrotic remodeling. These processes interact bidirectionally with mitochondrial dysfunction and ROS generation, creating a self-reinforcing cycle of oxidative injury and structural deterioration[16,23]. Although ERK and PI3K-Akt-mTOR pathways partially counterbalance these processes, persistent inflammatory activation may overwhelm endogenous repair signaling in the absence of external modulation[2,9].

Situating exercise conditioning and MSC-EXO therapy within this inflammatory context reveals their broader mechanistic rationale. By reinforcing ERK and PI3K-Akt-mTOR survival signaling upstream of fibroblast activation and oxidative amplification, these interventions interrupt the self-reinforcing cycle of ROS generation and extracellular matrix deposition that otherwise drives progressive structural deterioration in ISO-induced myocardial injury.

Current clinical, translational, and preclinical evidence for MSC-based cardiac regeneration

The current literature spans randomized controlled trials, translational human investigations, preclinical animal studies, experimental delivery platforms, and narrative syntheses[2,6,7]. Two randomized controlled trials have evaluated intracoronary MSC therapy in the setting of acute myocardial infarction[12,13]. These trials demonstrate procedural feasibility and acceptable safety profiles, with modest signals of improvement in functional outcomes and attenuation of adverse ventricular remodeling in selected patient populations[12,13,19,26,27]. However, the magnitude and consistency of observed benefit remain variable, which highlights persistent challenges related to patient selection, dosing, delivery route, and endpoint sensitivity[12,13,17,19,28]. These trials do not provide definitive evidence of durable structural myocardial regeneration, which reinforces the need for mechanistic refinement[19,22].

Beyond randomized trials, the literature contains multiple non-randomized human translational and clinical analyses that focus on delivery feasibility, retention efficiency, and comparative administration routes[5,16,17,18]. They find that therapeutic efficacy is constrained less by cellular viability and more by biological engagement within the injured myocardial environment[2,16,17]. The research emphasizes limited cell retention, heterogeneous tissue integration, and variable paracrine signaling as major determinants of outcome[16,17]. As such, these investigations provide context for understanding why clinical efficacy has remained inconsistent despite encouraging safety profiles[2,17].

In contrast, preclinical animal studies constitute a substantial and mechanistically informative segment of the literature[2,8,11]. Multiple ischemic and ischemia–reperfusion models show that experimental interventions consistently demonstrate improvements in ventricular function, attenuation of adverse remodeling, enhanced angiogenesis, and reduced cardiomyocyte apoptosis[2,8,11]. These effects are reproducibly associated with activation of intracellular survival pathways, modulation of metabolic stress responses, and improved mitochondrial integrity[2,8]. Importantly, several animal studies explicitly demonstrate that therapeutic benefit is mediated predominantly through paracrine mechanisms rather than durable engraftment, providing biological plausibility for cell-free strategies[2,6,7].

In parallel, experimental and bioengineering studies have focused on optimizing therapeutic delivery and signal potency[10,14,21]. Engineered exosome platforms, ultrasound-triggered targeting systems, biomimetic nanoparticles, and hydrogel-based retention strategies consistently enhance tissue targeting, prolong biological activity, and amplify downstream signaling effects in preclinical models[10,14,21]. Historically, intracellular delivery of therapeutic vesicles and nanoparticles has relied predominantly on endocytosis, whereby extracellular cargo is internalized into early endosomes and subsequently trafficked through late endosomal and lysosomal compartments before potential cytosolic release[10]. While this pathway enables cellular uptake, it introduces inherent biological limitations, including delayed intracellular availability, enzymatic degradation, and signal attenuation due to vesicular sequestration, which can substantially reduce effective payload bioactivity despite successful internalization[10]. As a result, a significant proportion of delivered proteins, RNAs, and signaling molecules may become degraded or mislocalized before engaging their intended intracellular targets[10]. Preclinical myocardial infarction models demonstrate that ultrasound-triggered exosome and nanoparticle delivery enhances myocardial accumulation, accelerates intracellular signaling activation, and improves functional recovery compared with delivery strategies that rely primarily on endocytosis[20,28]. These studies directly address limitations identified in clinical trials and provide mechanistic justification for transitioning from bulk cell delivery toward precision-guided, paracrine-focused interventions[2,17].

The narrative and mechanistic review literature, which represents the largest component of the current body of work finds that MSC-based therapies exert their effects primarily through secreted factors, including extracellular vesicles and exosomes, which modulate cardiomyocyte survival, angiogenesis, inflammatory signaling, and remodeling dynamics[2,6,7]. These also include editorial and commentary perspectives that critically assess the reproducibility and clinical significance of intracoronary cell therapy trials[19,22]. Rather than undermining the field, these critiques reinforce the necessity of mechanistic clarity, rigorous delivery optimization, and biologically informed trial design[19,22].

The current body of literature supports several key conclusions (Table 1)[2,6,7]. First, regenerative therapies in IHD are safe and biologically active but exhibit heterogeneous clinical efficacy[12,13,17,22]. Second, preclinical and experimental evidence strongly implicates paracrine and exosome-mediated signaling as the main drivers of observed benefit[2,6,8,10]. Third, delivery efficiency and biological engagement remain dominant barriers to translation[2,16,17]. Finally, the convergence of mechanistic insights from exercise biology, MSC signaling, and engineered paracrine platforms provides a rational framework for integrative cardioprotective strategies that might go beyond acute ischemic injury mitigation[1,2,10,14].

Safety and limitations

MSC-based and paracrine-focused regenerative strategies demonstrate a favorable short-term safety profile in both clinical and preclinical settings[2,12,13,17,19,26,27]. Randomized clinical trials evaluating intracoronary MSC administration after acute myocardial infarction consistently report acceptable procedural safety, with low rates of serious adverse events attributable to the intervention itself[12,13]. Similarly, translational human studies and delivery-focused investigations do not identify excess arrhythmic risk, microvascular obstruction, or clinically significant immune reactions when contemporary dosing and delivery protocols are employed[5,17,18,26,27].

There are also several important limitations that constrain therapeutic efficacy and reproducibility, foremost among these is variable biological engagement following cell delivery. Multiple reviews and delivery-route analyses emphasize limited myocardial retention, rapid washout, and inconsistent paracrine signaling as major barriers to durable benefit[2,16,17]. These factors likely contribute to the modest and heterogeneous functional improvements observed in clinical trials, despite robust preclinical efficacy[2,17,22].

A second limitation concerns context-dependent responsiveness of the ischemic myocardium[8,9]. Experimental and animal studies demonstrate that metabolic state, inflammatory milieu, and substrate availability influence therapeutic response, with evidence that factors such as altered amino acid metabolism or advanced myocardial aging can restrict regenerative efficacy[8,9]. These findings could suggest that regenerative interventions do not operate in isolation but interact with complex host biology that is incompletely captured in early-phase clinical trial designs[2,9].

Additionally, although paracrine and exosome-mediated mechanisms are emphasized, standardization of exosome composition, dosing, and bioactivity remains lacking[7,10,14]. Experimental studies demonstrate potent biological effects of engineered or targeted exosome platforms, but variability in production methods, cargo content, and delivery strategies complicates cross-study comparison and clinical translation[7,10,14]. Long-term safety data for repeated or high-dose paracrine delivery also remain limited[7].

Finally, critical commentary within the literature highlights concerns regarding trial design, endpoint selection, and interpretive overreach, particularly when modest surrogate improvements are extrapolated to broad clinical benefit[19,22]. These critiques reinforce the need for mechanistic alignment between intervention, biological target, and outcome measures, rather than reliance on infarct size or short-term functional metrics alone[19,22].

CONCLUSION

The evidence synthesized in this narrative review support a unifying mechanistic model in which cardioprotection in ISO-induced myocardial injury can be understood through convergent intracellular survival signaling centered on ERK and PI3K-Akt-mTOR networks[2,8,9]. Exercise conditioning promotes a phenotype of enhanced signaling responsiveness, regulated autophagy, and improved mitochondrial quality control, thereby increasing tolerance to catecholaminergic stress[1,6,7]. MSC-EXO reinforce these same survival pathways through paracrine delivery of regulatory microRNAs, heat shock proteins, antioxidative enzymes, and adaptor proteins that support angiogenesis, suppress apoptosis, and preserve mitochondrial function[6,10,14]. Translational work increasingly indicates that delivery efficiency and biological engagement are dominant determinants of therapeutic effect, motivating targeted, ultrasound-enabled, and nanoparticle-based platforms designed to improve myocardial retention and amplify downstream signaling (Table 1)[2,10,14,17]. Future investigations should enforce mechanistic alignment between intervention design, pathway engagement, and outcome selection, as the translation of preclinical biological plausibility into reproducible, durable clinical benefit in IHD depends fundamentally on the precision with which each intervention is matched to its intended molecular target and measured by endpoints sensitive to the specific reparative process being engaged[19,22].