Excessive mitochondrial fission in myocardial ischemia/reperfusion injury: Pathophysiological relevance and potential cardioprotective modulation by plant-derived interventions and electroacupuncture

Abstract

Myocardial ischemia/reperfusion injury (MI/RI) remains an unresolved problem despite reperfusion therapies, including percutaneous coronary intervention. Mitochondria are central mediators of reperfusion-associated cardiomyocyte injury, with excessive fission implicated as a potentially important mechanism linking acute metabolic stress to fragmentation, bioenergetic failure, and cell death. Reactive oxygen species, Ca²⁺ overload, mitochondrial permeability transition pore opening and membrane-potential loss promote dynamin-related protein 1-centered (Drp1-centered) fission, often involving mitochondrial fission factor, mitochondrial fission 1 protein and site-specific Drp1 phosphorylation. By disrupting mitochondrial networks and oxidative phosphorylation, this process contributes to cytochrome c release, apoptosis, microvascular dysfunction and impaired recovery. Impaired mitofusin 1/2-mediated and optic atrophy 1-mediated fusion limits repair and quality control, supporting investigation of this fission-dominant imbalance as a target for cardioprotection. Preclinical studies suggest plant-derived natural products, multi-component botanical preparations and electroacupuncture may influence this imbalance. Reported effects include attenuated Drp1 signaling, altered fusion-protein expression, preserved membrane potential, inhibited permeability transition, reduced oxidative stress and apoptosis, and modulated upstream metabolic/stress-responsive pathways. However, hypoxia/reoxygenation, oxygen-glucose deprivation/reoxygenation, non-reperfused ischemia/infarction and stress models provide surrogate or supportive evidence rather than direct evidence of MI/RI. Heterogeneity in models, timing, dosage, readouts, causal validation and botanical quality control limits interpretation. This review examines these mechanisms, interventions and translational priorities. Clinical efficacy and safety of these interventions in MI/RI have not been established.

Key Words: Myocardial ischemia/reperfusion injury; Mitochondrial fusion; Mitochondrial fission; Dynamin-related protein 1; Optic atrophy 1; Mitochondrial fragmentation; Cardioprotection; Plant-derived natural products; Electroacupuncture

Core Tip: Excessive mitochondrial fission is not merely a morphological consequence of myocardial ischemia/reperfusion injury, but a potential amplifier of mitochondrial dysfunction and cardiomyocyte death. This review highlights the complementary contributions of dynamin-related protein 1-centered fission activation and impaired fusion-mediated network repair. It further emphasizes that broad mitochondrial protection should not be equated with direct modulation of the fusion-fission machinery. Plant-derived interventions and electroacupuncture show experimental promise, but mechanistic causality and translational relevance remain insufficiently established.

INTRODUCTION

Timely reperfusion is the most effective strategy for limiting myocardial necrosis in patients with acute myocardial infarction. Reperfusion therapies, particularly percutaneous coronary intervention, have substantially improved patient survival and cardiac outcomes. Nevertheless, the abrupt restoration of blood flow can paradoxically induce additional myocardial damage, a process known as myocardial ischemia/reperfusion injury (MI/RI)[1,2]. This injury contributes to cardiomyocyte death, microvascular dysfunction, arrhythmia, adverse ventricular remodeling and incomplete functional recovery after reperfusion[3]. Despite extensive investigation, effective treatments specifically targeting reperfusion-associated myocardial injury remain limited, underscoring the need to identify tractable molecular processes linking reperfusion stress to myocardial damage.

Mitochondria are central mediators of MI/RI because cardiomyocytes depend heavily on mitochondrial oxidative phosphorylation to sustain contractile function[4]. During ischemia and early reperfusion, abrupt changes in oxygen availability, substrate metabolism and ionic homeostasis promote excessive reactive oxygen species (ROS) generation, Ca²⁺ overload, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (ΔΨm) dissipation. These disturbances impair adenosine triphosphate (ATP) production, facilitate cytochrome c release and activate cell-death signaling[5]. Structurally, MI/RI is frequently accompanied by pronounced mitochondrial fragmentation, with elongated or interconnected mitochondrial networks becoming shorter, punctate, and structurally disrupted. This fragmented phenotype provides a morphological link between acute reperfusion stress and mitochondrial dysfunction.

However, mitochondrial fragmentation should not be attributed to fission alone. Mitochondria continuously undergo fission and fusion, and their morphology reflects the relative activity of these opposing processes. Under physiological conditions, fission supports mitochondrial distribution, segregation of damaged components and quality control, whereas fusion facilitates content exchange, functional complementation and maintenance of network integrity. Accordingly, the fragmented mitochondrial phenotype observed after MI/RI may result from enhanced fission, impaired fusion or both. Available evidence indicates that dynamin-related protein 1-centered (Drp1-centered) excessive mitochondrial fission is a major contributor to mitochondrial fragmentation and myocardial injury during MI/RI[5,6]. Drp1 is recruited from the cytosol to the outer mitochondrial membrane (OMM) through interactions with several mammalian receptor or adaptor proteins, including mitochondrial fission factor (Mff), mitochondrial fission 1 protein (Fis1), and mitochondrial dynamics protein 49 (MiD49) kDa and MiD51 kDa, although their relative contributions are context-dependent. Recruited Drp1 then assembles at prospective division sites to drive membrane constriction and scission[7]. Meanwhile, reduced or disrupted mitofusin 1/2-mediated (MFN1/2-mediated) and optic atrophy 1-mediated (OPA1-mediated) fusion limits network restoration and further sustains mitochondrial fragmentation. MI/RI-associated mitochondrial injury can therefore be understood as a fission-dominant fusion-fission imbalance, in which excessive fission predominates and impaired fusion amplifies network disruption and cardiomyocyte injury.

Plant-derived natural products, multi-component botanical preparations and electroacupuncture have increasingly been investigated as potential modulators of this pathological process. Many of these interventions derive from traditional medicine practices, but their cardioprotective effects can be evaluated within modern pharmacological and cardiovascular research frameworks.

Recent reviews have synthesized relevant evidence from complementary perspectives: Pan et al[8] examined mitophagy, mitochondrial dynamics and biogenesis as targets of traditional Chinese medicine across cardiovascular diseases, whereas Zhang et al[9] reviewed Chinese herbal medicine in MI/RI across energy metabolism, mitochondrial quality control, oxidative stress and Ca²⁺ regulation. The present review provides a focused appraisal of excessive mitochondrial fission, distinguishing fission-specific mechanistic evidence from broader mitochondrial protection and comparing model directness, causal validation and intervention timing. Studies in this literature describe changes in Drp1-related, MFN1/2-related and OPA1-related markers, reduced mitochondrial fragmentation, preserved ΔΨm, inhibited mitochondrial permeability transition and attenuated oxidative stress and cardiomyocyte apoptosis. However, the evidence remains dispersed across intervention categories, experimental models, treatment schedules, molecular readouts and signaling pathways. Building on these reviews while taking a distinct approach, the present review organizes the evidence along four dimensions: (1) Drp1-centered excessive fission, impaired fusion-mediated network repair, or broader mitochondrial protection; (2) Model directness, distinguishing direct in vivo myocardial I/R, ex vivo isolated-heart I/R, cellular hypoxia/reoxygenation (H/R) or oxygen-glucose deprivation/reoxygenation (OGD/R), permanent ischemia or MI without reperfusion, and other indirect injury models; (3) Mechanistic causality, ranging from protein-expression changes to assessments of mitochondrial morphology or protein translocation and to target manipulation; and (4) Intervention timing and its relevance to potential clinical treatment windows, distinguishing pretreatment from administration during ischemia, at reperfusion onset or after reperfusion. Model directness, mechanistic causality and intervention timing represent complementary but independent dimensions of evidence and should not be conflated. Within this framework, much of the available evidence remains associative or derives from surrogate or indirect injury models, while direct causal validation in myocardial I/R models remains limited. Therefore, this review focuses on excessive mitochondrial fission as a potentially important pathophysiological process contributing to mitochondrial fragmentation in MI/RI, while considering impaired fusion as an interacting process that exacerbates and sustains mitochondrial network disruption. We examine the physiological machinery, reperfusion-associated disturbances and intervention evidence, then discuss unresolved mechanisms and translational priorities.

LITERATURE SEARCH AND STUDY SELECTION

For this narrative review, PubMed and the China National Knowledge Infrastructure (CNKI) were searched. The search covered database inception through December 31, 2025, the final search date, with no language restrictions. Representative search concepts included myocardial ischemia/reperfusion or MI/RI; mitochondrial fission, fusion or dynamics; Drp1, MFN1, MFN2 or OPA1; and the names of plant-derived compounds, botanical formulations or electroacupuncture. The core PubMed query was: ((“mitochondrial fission”[Title/Abstract]) OR (“mitochondrial fusion”[Title/Abstract])) AND (“myocardial ischemia reperfusion injury”[Title/Abstract]). Targeted supplementary searches combined disease and mitochondrial concepts with intervention names identified during screening. Equivalent Chinese-language terms and Boolean logic were used in CNKI, and the reference lists of relevant articles were screened. Mechanistic studies were prioritized according to direct relevance to MI/RI, particularly those assessing mitochondrial morphology, protein translocation, target manipulation or functional outcomes. For detailed synthesis in Tables 1 and 2, 22 representative original intervention studies evaluating plant-derived compounds, botanical formulations or electroacupuncture were selected on the basis of model relevance, mechanistic specificity, and completeness of intervention and outcome reporting. Duplicate records, reviews, irrelevant reports and studies without relevant fusion-fission-related outcomes were excluded from detailed synthesis. The extracted information comprised species and model, selected study groups, dose, route, administration timing, endpoint-specific sample sizes, randomization and blinding status, representative cardiac outcomes and the use of inhibitors, knockdown or other causal approaches; a second reviewer independently verified all extracted information against the original reports. Numerical outcomes were transcribed only when explicitly reported in source text, tables, figure labels or supplementary material; values were not estimated from graphs. Because this was a narrative rather than systematic synthesis, model directness, mechanistic causality and clinical timing relevance were evaluated as complementary but independent dimensions.

Table 1 Chemically defined plant-derived compounds associated with mitochondrial fusion-fission regulation in myocardial ischemia/reperfusion injury and related experimental models.

Compound	Chemical class	Major plant source	Experimental model	Study groups	Sample size and design	Dosage, administration route, and regimen	Main findings	Evidence strength	Ref.
Ginsenoside Rg1	Triterpenoid saponin	Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma)	H9c2 H/R (3 hours/3 hours)	Ctrl; H/R; H/R + Rg1	3 independent experiments; R/B: NR	3.125, 6.25, 12.5 μmol/L; MEC NR; pretreated for 24 hours before H/R	Cell viability 66.86% ± 2.655% vs 54.39% ± 0.6338% (H/R; 12.5 μmol/L; mean ± SEM). ↑: MFN2, GDH activity, intracellular glutamate, cell viability, ATP, ΔΨm. ↓: Mitochondrial fragmentation, LDH, CK, ROS, Bax/Bcl-2	C; MFN2 siRNA; Pre (C): Clinical window untested	Dong et al[53]
Ginsenoside Rg1	Triterpenoid saponin	Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma)	HL-1 H/R (6 hours/2 hours)	Ctrl; H/R; H/R + Rg1; H/R + Rg1 + siNotch1	n = 6 (viability/LDH), 3 (protein assays); R/B: NR	200 μmol/L; MEC NR; added at hypoxia onset and maintained throughout H/R	↑: Notch1, MFN1, ΔΨm, ATP, cell viability. ↓: MFN2, LDH, cytosolic ROS	C; Notch1 siRNA; Isch (C): Clinical window untested	Zhang et al[54]
Ginsenoside Rg5	Triterpenoid saponin	Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma)	ICR mice, ISO-induced ischemic injury; NRVMs challenged with PA (100 μmol/L, 2 hours), with or without subsequent H/R (4 hours/1 hour)	In vivo: Sham; ISO; ISO + Rg5; TMZ + ISO. In vitro: Ctrl; PA; Rg5 + PA; H/R; PA + H/R; Rg5 + PA + H/R	In vivo: n = 4 (localization), 3-6 (TUNEL). Cells: 3 independent experiments; R/B: NR	In vivo: 50 mg/kg; MED NR; oral; administered 1 hour before ISO on 2 consecutive days. In vitro: 10 μmol/L; MEC NR; pretreated for 0.5 hours before PA	In vivo: ↑: Mitochondrial HK II, and Akt; ↓: Mitochondrial Drp1 recruitment. In vitro: PA model: ↑: P-Akt, mitochondrial HK II, p-Drp1(Ser637), ΔΨm; ↓: Mitochondrial Drp1 recruitment, mPTP opening, ROS, mitochondrial fragmentation. PA + H/R model: ↑: ATP; ↓: Cyt c release, ROS, cell death	C, E; Akt/Drp1 siRNA (cells); Pre: Post-onset untested	Yang et al[55]
Ginsenoside Rc	Triterpenoid saponin	Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma)	SD rat LAD I/R (2 hours/6 hours); H9c2 H/R (time NR)	In vivo: Sham; I/R; Rc + I/R. In vitro: Ctrl; H/R; Rc + H/R	In vivo: n = 5 (infarct); R: Yes; B: NR	In vivo: 10 mg/kg; MED NR; administered for 14 days before I/R; route and frequency NR. In vitro: 2.5 μmol/L, 5 μmol/L, 10 μmol/L; MEC NR; pretreated for 48 hours before H/R	In vivo: ↑: SIRT1, ETC complex III/IV activities; ↓: Infarct size. In vitro: ↑: SIRT1, ETC complex I-III protein levels, ATP, Bcl-2/Bax; ↓: PGC-1α acetylation, Cyt c	A, C; SIRT1 perturbation (cells); Pre: Post-onset untested	Huang et al[56]
Calenduloside E	Triterpenoid saponin	Aralia elata (Miq) Seem (Araliaceae; NR)	SD rat LAD I/R (30 minutes/48 hours); H9c2 H/R (6 hours/reoxygenation duration NR)	In vivo: Sham, Sham + CE, I/R, I/R + CE. In vitro: Ctrl, H/R, H/R + CE	In vivo: n = 6 (cardiac). Cells: 3 independent experiments; R: Yes (rats); B: NR	In vivo: 15 mg/kg/day; MED NR; gavage for 3 days; last dose 60 minutes before ligation. In vitro: 1 μmol/L, 2 μmol/L, 4 μmol/L, 8 μmol/L; MEC NR; pretreated for 4 hours before hypoxia	In vivo: ↑: MFN2, OPA1, p-AMPK/AMPK, ATP, EF, FS; ↓: Drp1, cTnI, infarct size, Cyt c release, mitochondrial Bax translocation. In vitro: ↑: MFN2, OPA1, p-AMPK/AMPK, ATP, cell viability; ↓: Drp1, Fis1, cleaved caspase-3, cleaved PARP, LDH	A, C; OPA1 siRNA/AMPK blockade (C); Pre: Post-onset untested	Wang et al[60]
Notoginsenoside R1	Triterpenoid saponin	Panax notoginseng (Burkill) F.H. Chen (Araliaceae; Notoginseng Radix et Rhizoma)	H9c2 H/R (6 hours/2 hours)	Ctrl; Ctrl + NGR1; H/R; H/R + NGR1	n = 8 (viability), 6 (LDH); R/B: NR	25 μmol/L; MEC NR; pretreated for 2 hours before H/R and maintained throughout H/R	↑: OPA1, MFN1/2, PGC-1α, NRF1, NRF2, ATP, ETC complexes I-V activity, cell viability. ↓: Drp1, Fis1, mPTP opening, ΔΨm depolarization, ROS, LDH, PINK1, Parkin, BNIP3	C; Assoc.; Pre (C): Clinical window untested	Xu et al[59]
Astragalosidic acid (LS-102)	Triterpenoid saponin derivative	As astragaloside IV derivative; parent compound from Astragalus membranaceus (Fisch) Bunge (Fabaceae; Astragali Radix)	SD rat LAD I/R (30 minutes/90 minutes); H9c2 H/R (12 hours/2 hours)	Sham; I/R; I/R + LS-102; positive control (verapamil). In vitro: Ctrl; H/R; H/R + LS-102	In vivo: n = 6 (infarct), 8-10 (arrhythmia); R: Yes; B: NR	In vivo: 2.5 mg/kg, 5 mg/kg, 10 mg/kg; MED NR; gavage; administered 30 minutes before LAD ligation. In vitro: 0.3125 μmol/L, 0.625 μmol/L, 1.25 μmol/L; MEC NR; pretreated for 24 hours before H/R	VT/VF incidence 10% (1/10) vs 88% (7/8) (I/R; 10 mg/kg). In vivo: ↑: P-PI3K, p-Akt; ↓: Infarct size, CK, LDH In vitro: ↑: P-Drp1(Ser637), p-PI3K, p-Akt, p-GSK-3β(Ser9), ATP, SOD, ΔΨm, Bcl-2/Bax ratio; ↓: P-Drp1(Ser616), CK, LDH, Ca2+ overload, ROS, Cyt c	A, C; PI3K/Akt blockade; Pre: Post-onset untested	Chen et al[58]
Vitexin	Flavonoid glycoside	Crataegus pinnatifida Bunge (Rosaceae; Crataegi Fructus)	SD rat Langendorff I/R (30 minutes/30 minutes); H9c2 H/R (5 hours/2 hours)	Shared (ex vivo/in vitro): Vehicle control; I/R (H/R); VT + I/R (H/R)	Ex vivo: n = 6 (cardiac); cells: n = 4 (protein); R: Yes (both); B: NR	Ex vivo: 10 μmol/L in perfusate; MED NR; perfused for 20 minutes before ischemia and during 30-minute reperfusion. In vitro: 10 μmol/L; MEC NR; pretreated for 2 hours before H/R	Ex vivo: ↑: MFN2, Bcl-2/Bax ratio; ↓: Mitochondrial Drp1, Epac1, Rap1, cytosolic Cyt c, cleaved caspase-3/9, infarct size. In vitro: ↑: MFN2, ΔΨm; ↓: Mitochondrial Drp1, ROS, Epac1, Rap1, NOX4, mitochondrial Bax, cleaved caspase-3/9	B, C; Epac agonist/inhibitor; Pre+Rep (B): Rep-only untested; Pre (C)	Yang et al[67]
Vitexin	Flavonoid glycoside	Crataegus pinnatifida Bunge (Rosaceae; Crataegi Fructus)	SD rat Langendorff I/R (30 minutes/30 minutes); H9c2 H/R (5 hours/1 hour)	Ex vivo: Ctrl; I/R; I/R + VT. In vitro: Ctrl; H/R; H/R + VT	Ex vivo: n = 5-6 (infarct), 6-7 (function); R: Yes (hearts/cells); B: NR	Ex vivo: 1 μmol/L, 3 μmol/L, 10 μmol/L in perfusate; MED NR; perfused for 20 minutes before I/R. In vitro: 1 μmol/L, 3 μmol/L, 10 μmol/L, 30 μmol/L; MEC NR; pretreated for 2 hours before H/R	Ex vivo: ↑: MFN2, COX IV, CF, Bcl-2/Bax ratio; ↓: Mitochondrial Drp1, NOX4, cytosolic Cyt c, cleaved caspase-3/9, infarct size, LDH. In vitro: ↑: MFN2, ATP, ΔΨm; ↓: Mitochondrial Drp1, LDH, cleaved caspase-3/9, ROS	B, C; Assoc; Pre: Post-onset untested	Xue et al[68]
Berberine	Isoquinoline alkaloid	Coptis chinensis Franch (Ranunculaceae; Coptidis Rhizoma)	SD rats ISO-induced injury; H9c2 CoCl2 hypoxia-mimetic injury	In vivo: Vehicle; ISO; ISO + BBR. In vitro: Ctrl; CoCl2; CoCl2 + BBR	In vivo: n = 6 (serum), 3 (protein); cells: n = 3; R: Yes (rats); B: NR	In vivo: 100 mg/kg/day; MED NR; gavage for 14 days before ISO. In vitro: 2.5 μmol/L; MEC NR; pretreated for 24 hours before CoCl2 exposure	In vivo: ↑: MFN2 (mRNA), ATP, Beclin 1, p62, p-AMPK, p-Akt, LKB1, p-Drp1/Drp1; ↓: LDH, CK. In vitro: ↑: Drp1, Beclin 1, p62, p-AMPK, p-Akt, LKB1; ↓: ROS, LDH	E; Assoc; Pre: Post-onset untested	Yang et al[71]
Gastrodin	Phenolic glycoside	Gastrodia elata Blume (Orchidaceae; Gastrodiae Rhizoma)	H9c2 H2O2 oxidative injury (400 μmol/L, 3 hours); additional I/R-like injury (9 hours/3 hours)	H2O2 model: Ctrl; H2O2; GAS + H2O2. I/R-like model: Ctrl; I/R-like; GAS + I/R-like	3 independent experiments; R/B: NR	20 μmol/L, 50 μmol/L, 100 μmol/L; MEC NR; pretreated for 4 hours before injury induction	H2O2 model: ↑: MFN2 and OPA1 mRNA; ↓: Fis1 mRNA, mitochondrial fragmentation, ROS. NRF2-siRNA blunted protection in the H2O2 model; GAS did not significantly alter Drp1 mRNA	C, E; NRF2 siRNA (E); Pre (C,E): Clinical window untested	Cheng et al[62]
Rhein	Anthracenecarboxylic acid	Rheum palmatum L (Polygonaceae; Rhei Radix et Rhizoma)	SD rat LAD-ligation AMI; H9c2 hypoxia 12 hours	Sham; AMI; AMI + rhein; AMI + mdivi-1. In vitro: Ctrl; hypoxia; hypoxia + rhein	In vivo: n = 5 (serum), 3 (TUNEL/protein); R: Yes; B: NR	In vivo: 90 mg/kg; MED NR; gavage for 8 weeks before AMI; frequency NR. In vitro: 1 μg/mL; MEC NR; pretreated for 1 hour before hypoxia	Myocardial TUNEL-positive cells ≈ 15% vs > 30% (AMI; author-reported approximations). Shared (in vivo/in vitro): ↑: OPA1, Beclin 1, PINK1, Parkin, LC3-II/LC3-I; ↓: Drp1, Bax/Bcl-2, cleaved caspase-3. In vivo only: ↓: CK, CK-MB, cTnT, LDH, infarct size	D, E; mdivi-1 comparator; Pre: Post-onset untested	Li et al[73]
Salidroside	Phenylethanoid glycoside	Rhodiola rosea L (Crassulaceae; Rhodiolae roseae Rhizoma et Radix)	SD rat LAD I/R (30 minutes/2 hours); H9c2 H/R (9 hours/6 hours)	In vivo: Sham; I/R; I/R + Sal; I/R + Sal + compound C. In vitro: Ctrl; H/R; H/R + Sal; H/R + Sal + AMPK siRNA	In vivo: n = 6 (infarct/function); R: Yes (rats/cells); B: NR	In vivo: 40 mg/kg; MED NR; administered i.v. 5 minutes before ischemia. In vitro: 10 μmol/L; MEC NR; pretreated for 30 minutes before hypoxia	In vivo: ↑: MFN2, OPA1, p-AMPK/AMPK, ATP, Bcl-2; ↓: P-Drp1(Ser616), Fis1, CHOP, infarct size, LDH, CK, cleaved caspase-3, Bax. In vitro: ↑: P-AMPK/AMPK, Bcl-2; ↓: P-Drp1(Ser616), LDH, CK, CHOP, cleaved caspase-3, Bax	A, C; AMPK blockade/siRNA (C); Pre: Post-onset untested	Tian et al[64]

Selected models, groups and findings are shown; ↑/↓ denote increases/decreases. Minimum effective dose/minimum effective concentration require dose-response support; otherwise not reported. Botanical names and medicinal parts follow authoritative databases and pharmacopoeial/European Medicines Agency monographs. Evidence strength summarizes model directness, mechanistic tests and timing relevance. Model categories: A: In vivo myocardial ischemia/reperfusion (I/R) in an intact animal; B: Isolated-heart I/R without intact systemic context; C: Cellular hypoxia/reoxygenation (R) or oxygen-glucose deprivation/R surrogates; D: Permanent myocardial ischemia/myocardial infarction (MI) without reperfusion; E: Other injury models. A/B directly model cardiac I/R; C models cellular components; D/E provide indirect evidence. Categories are not overall evidence-quality ranks. Assoc: No targeted causal test; named perturbations support the tested pathway, not necessarily fission mediation; comparators do not establish necessity. Pre: Before injury (post-onset initiation untested); Isch: During ischemia; Rep: At/after reperfusion onset. Acute-treatment relevance depends on start time, model, route and exposure; Pre + Rep does not establish Rep-only efficacy. Post-MI: After non-reperfused MI (post-infarction recovery). Cellular timing does not establish clinical windows; timing relevance does not establish clinical efficacy. Sample sizes are endpoint-specific and per group unless independent experiments are specified; cellular n retains the reported replication unit. Only explicitly reported outcome values are shown; graph estimates are excluded. ≈ denotes an approximation stated by the source authors; dispersion is retained as reported. R: Randomization; B: Reported blinding stage; yes: Reported; NR: Not reported; H/R: Hypoxia/reoxygenation; SEM: Standard error of the mean; MFN1/2: Mitofusin 1/2; GDH: Glutamate dehydrogenase; ATP: Adenosine triphosphate; ΔΨm: Mitochondrial membrane potential; LDH: Lactate dehydrogenase; CK: Creatine kinase; ROS: Reactive oxygen species; Bax/Bcl-2: Bcl-2-associated X protein to B-cell lymphoma 2; ISO: Isoproterenol; NRVMs: Neonatal rat ventricular myocytes; PA: Palmitic acid; TMZ: Trimetazidine; HK II: Hexokinase II; Akt: Protein kinase B; Drp1: Dynamin-related protein 1; mPTP: Mitochondrial permeability transition pore; Cyt c: Cytochrome c; LAD: Left anterior descending coronary artery; I/R: Ischemia/reperfusion; SIRT1: Sirtuin 1; ETC: Electron transport chain; PGC-1α: Peroxisome proliferator-activated receptor-γ coactivator 1α; CE: Calenduloside E; OPA1: Optic atrophy 1; AMPK: AMP-activated protein kinase; EF: Ejection fraction; FS: Fractional shortening; cTnI: Cardiac troponin I; PARP: Poly(adenosine diphosphate-ribose) polymerase; NGR1: Notoginsenoside R1; NRF1: Nuclear respiratory factor 1; NRF2: Nuclear factor erythroid 2-related factor 2; PINK1: Phosphatase and tensin homolog-induced kinase 1; BNIP3: B-cell lymphoma 2/adenovirus early region 1B 19-kDa-interacting protein 3; VT/VF: Ventricular tachycardia/ventricular fibrillation; PI3K: Phosphoinositide 3-kinase; GSK-3β: Glycogen synthase kinase 3β; SOD: Superoxide dismutase; Epac1: Exchange protein directly activated by cyclic adenosine monophosphate 1; NOX4: NADPH oxidase 4; BBR: Berberine; LKB1: Liver kinase B1; GAS: Gastrodin; AMI: Acute myocardial infarction; LC3-II: Light chain 3-II; CK-MB: Creatine kinase-MB; cTnT: Cardiac troponin T; Fis1: Mitochondrial fission 1 protein; CHOP: CCAAT/enhancer-binding protein homologous protein.

Table 2 Experimental evidence for plant-derived interventions and electroacupuncture associated with mitochondrial fusion-fission-related changes in myocardial ischemia/reperfusion injury and related experimental models.

Intervention type	Intervention	Composition or acupoints	Experimental model	Study groups	Sample size and design	Dosage, administration route, and regimen	Main findings	Evidence strength	Ref.
Defined combination	GRS	Ginsenoside Rb1 + ruscogenin + schisandrin (ratio: 6:0.75:6)	In vivo: ICR mice LAD I/R (30 minutes/24 hours). In vitro: H9c2 H/R (6 hours/6 hours; 2-hour reoxygenation for Drp1 and 1-hour for AMPK assays); NRVMs H/R (12 hours/12 hours)	In vivo: Sham; I/R; GRS + I/R; positive control (captopril). In vitro: Ctrl; H/R; GRS + H/R	In vivo: n = 6 (cardiac); R: Yes; B: Surgery	In vivo: 6.4 mg/kg, 12.8 mg/kg, 19.2 mg/kg; MED NR; administered i.p. within 10 minutes after reperfusion onset. In vitro: 0.1 μg/mL, 1 μg/mL, 10 μg/mL; MEC NR; added at hypoxia onset and maintained throughout H/R	In vivo: ↑: EF; ↓: Infarct size. In vivo/in vitro: ↑: Bcl-2/Bax ratio; ↓: P-Drp1(Ser616), LDH, caspase-3. In vivo/in vitro: ↑: P-AMPK	A, C; AMPK siRNA (C); Early Rep (A): Potential acute relevance; Isch (C)	Li et al[80]
PBM (injection)	Shenmai Injection	Aqueous extracts from Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma) and Ophiopogon japonicus (Thunb) Ker Gawl (Asparagaceae; Ophiopogonis Radix)	In vivo: SD rat MI model (LAD ligation). In vitro: H9c2 H/R (12 hours/2 hours)	In vivo: Sham; MI; MI + SMI; positive control (valsartan). In vitro: Ctrl; H/R; SMI + H/R	In vivo: n = 8 (EF/FS), 3 (infarct); R: Yes; B: Echocardiography	In vivo: 0.38 mL/kg; MED NR; post-MI treatment; SMI route/frequency NR (28-day protocol). In vitro: 1 μL/mL, 2.5 μL/mL, 5 μL/mL; MEC NR; pretreated for 24 hours before H/R	In vivo: ↑: EF, FS; ↓: Infarct size. In vitro: ↑: MFN1/2 and OPA1 mRNA, Parkin, Beclin 1, LC3-II/LC3-I, ΔΨm; ↓: Drp1 and Fis1 mRNA, mitochondrial fragmentation, cytosolic Ca2+ overload, mPTP opening	C, D; Assoc; Post-MI (D): Recovery; Pre (C)	Yu et al[74]
PBM (injection)	Guanxinning Injection	Extracts of Salvia miltiorrhiza Bunge (Lamiaceae; Salviae Miltiorrhizae Radix et Rhizoma) and Ligusticum chuanxiong Hort (Apiaceae; Chuanxiong Rhizoma)	Wistar rat LAD I/R (30 minutes/60 minutes) in a CRF background	Sham; CRF; I/R; CRF + I/R + IPost; CRF + I/R + IPost + GXN; CRF + I/R	n = 15 (allocated); R: Yes; B: NR	Administered i.v. 10 minutes after ischemia onset; dose NR; MED NR	AN/AAR: 0.17 ± 0.09 (GXN + IPost), 0.39 ± 0.07 (CRF + I/R), 0.29 ± 0.07 (IPost); mean ± SD. GXN + IPost vs CRF + I/R: ↑: MFN1, OPA1; ↓: Drp1, cTnT, CK-MB. GXN + IPost vs IPost alone: ↑: MFN1, OPA1; ↓: CK-MB	A; Assoc; Isch + IPost: Potential acute relevance (combined)	Wang et al[75]
PBM (capsule)	Shuangshen Ningxin Formula	Extracts of Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma) + Salvia miltiorrhiza Bunge (Lamiaceae; Salviae Miltiorrhizae Radix et Rhizoma) + Corydalis yanhusuo W.T.Wang ex Z.Y.Su & C.Y.Wu (Papaveraceae; Corydalis Rhizoma)	In vivo: SD rat LAD I/R (45 minutes/24 hours). In vitro: CMECs OGD/R (4 hours/2 hours)	In vivo: Sham; I/R; SSNX + I/R. In vitro: Ctrl; OGD/R; SSNX + OGD/R	In vivo: n = 6 (EF); cells: n = 6 (viability); R: Yes (rats); B: NR	In vivo: 45 mg/kg/day, 90 mg/kg/day, 180 mg/kg/day; MED NR; gavage for 7 days before I/R. In vitro: 25 μg/mL; MEC NR; added before OGD; lead time NR	In vivo: ↑: EF. In vivo/in vitro: ↓: NR4A1, Mff, Drp1, HK II, Cyt c, caspase-9	A, C; NR4A1 inhibitor comparator (C); Pre: Post-onset untested	Liu et al[83]
Botanical decoction	Taohong Siwu Decoction	Prunus persica (L) Batsch (Rosaceae; Persicae Semen) + Carthamus tinctorius L (Asteraceae; Carthami Flos) + Angelica sinensis (Oliv) Diels (Apiaceae; Angelicae Sinensis Radix) + Paeonia lactiflora Pall (Paeoniaceae; Paeoniae Radix Alba) + Ligusticum chuanxiong Hort (Apiaceae; Chuanxiong Rhizoma) + Rehmannia glutinosa (Gaertn) Libosch ex DC (Orobanchaceae; Rehmanniae Radix Praeparata)	SD rat MI (LAD ligation)	MI; THSWD + MI	n = 13 (allocated); R: Yes; B: Echocardiography	1 mL/day; MED NR; gavage for 4 weeks, starting 2 days after MI (reported concentration 1.13 g/mL)	4-week: ↓ LVESV; EF/FS differences NS. ↑: P-Akt/Akt. ↓: Fis1, mitochondrial ROS; LC3B: No statistically significant change	D; Assoc; Post-MI: Recovery	Luo et al[82]
PBM (pill)	Qishen Yiqi Dripping Pill	Astragalus membranaceus (Fisch) Bunge (Fabaceae; Astragali Radix) + Salvia miltiorrhiza Bunge (Lamiaceae; Salviae Miltiorrhizae Radix et Rhizoma) + Dalbergia odorifera T.C.Chen (Fabaceae; Dalbergiae Odoriferae Lignum) + Panax notoginseng (Burkill) F.H. Chen (Araliaceae; Notoginseng Radix et Rhizoma)	ICR male mice MI (LAD ligation)	Sham; MI; QSYQ + MI; positive control (enalapril)	n = 6 (LVEF); R: Yes; B: H&E assessment	12 mg/kg/day, 35 mg/kg/day, 105 mg/kg/day; MED NR; gavage for 14 days after MI	↑: LVEF, MFN2, L-OPA1/S-OPA1, PGC-1α, TFAM, NRF1. ↓: P-Drp1(Ser616)/Drp1	D; Assoc; Post-MI: Recovery	Wu et al[79]
PBM (capsule)	Danqi Soft Capsule	50% Panax notoginseng (Burkill) F.H. Chen (Araliaceae; Notoginseng Radix et Rhizoma) + 50% Salvia miltiorrhiza Bunge (Lamiaceae; Salviae Miltiorrhizae Radix et Rhizoma)	In vivo: SD rat LAD I/R (30 minutes/24 hours). In vitro: H9c2 H2O2 injury	In vivo: Sham; I/R; DQ + I/R. In vitro: Ctrl; H2O2; DQ + H2O2	In vivo: n = 4 (infarct), 6 (serum); R: Yes; B: Histology scoring	In vivo: 0.6 g/kg/day, 1.2 g/kg/day; MED NR; gavage for 5 days before I/R. In vitro: 300 mg/mL, 600 mg/mL; MEC NR; pretreated for 2.5 hours before H2O2 exposure	In vivo: ↑: Bcl-2; ↓: Infarct size, p-CaMKII, p-Drp1(Ser616), cTnT, LDH, cleaved caspase-3, Bax. In vitro: ↑: Bcl-2, ΔΨm; ↓: P-CaMKII, p-Drp1(Ser616), ROS, Cyt c, cleaved caspase-3, caspase-3/8/9 activities, Bax, mPTP opening	A, E; Assoc; Pre: Post-onset untested	Yang et al[76]
PBM (pill)	Tongmai Yangxin Pill	Multiple constituents, including Rehmannia glutinosa (Gaertn.) Libosch ex DC (Orobanchaceae; Rehmanniae Radix) + Spatholobus suberectus Dunn (Fabaceae; Spatholobi Caulis) + Ophiopogon japonicus (Thunb) Ker Gawl (Asparagaceae; Ophiopogonis Radix) + Glycyrrhiza uralensis Fisch ex DC (Fabaceae; Glycyrrhizae Radix et Rhizoma) + Reynoutria multiflora (Thunb) Moldenke (Polygonaceae; Polygoni Multiflori Radix Praeparata) + Schisandra chinensis (Turcz) Baill (Schisandraceae; Schisandrae Chinensis Fructus) + Codonopsis pilosula (Franch) Nannf (Campanulaceae; Codonopsis Radix) + Ziziphus jujuba Mill (Rhamnaceae; Jujubae Fructus) + Cinnamomum cassia (L) J.Presl (Lauraceae; Cinnamomi Ramulus) + Asini Corii Colla + Mauremydis Carapax et Plastrum	In vivo: SD rat LAD I/R (30 minutes/120 minutes). In vitro: H9c2 H/R (6 hours/24 hours)	In vivo: Sham; I/R; TMYX + I/R (1/2/4 g/kg); positive control (raloxifene). In vitro: Ctrl; H/R; TMYX + H/R	In vivo: n = 9 (EF), 3 (infarct), 8 (serum); R: Yes; B: NR	In vivo: 1 g/kg/day, 2 g/kg/day, 4 g/kg/day; MED NR; gavage for 7 days after I/R establishment per Methods; pretreatment wording elsewhere is inconsistent. In vitro: 100 μg/mL; MEC NR; timing NR	In vivo: ↑: EF, MFN1/2; ↓: Drp1, Fis1, ROS, CK-MB, cTnI, LDH. In vitro: ↑: ERα, PGC-1α, ATP, ΔΨm; ↓: ROS, Bax and caspase-3 mRNA	A, C; ERα siRNA (C); timing inconsistent (A): Relevance unclear; NR (C)	Yu et al[84]
Electroacupuncture	Electroacupuncture pretreatment	Acupoints: PC6 + ST36 + CV4	SD rat LAD I/R (30 minutes/120 minutes)	Sham; I/R; EA + I/R; EA + compound C + I/R; EA + ML385 + I/R	n = 3 (ROS/SOD/MDA), 6 (protein); R: Yes; B: NR	EA: 20 minutes/day for 7 days before I/R (2 Hz, 1 mA); inhibitors administered i.p. 30 minutes before model induction	Cardiac function and infarct size were not assessed. ↑: SOD, AMPK, NRF2. ↓: Drp1, Mff, Fis1, ROS, MDA, mitochondrial structural damage	A; AMPK/NRF2 blockade; Pre: Post-onset untested	Zhang et al[86]

Selected models, groups and findings are shown; ↑/↓ denote increases/decreases. Minimum effective dose/minimum effective concentration require dose-response support; otherwise not reported. Botanical names and medicinal parts follow authoritative databases and pharmacopoeial/European Medicines Agency monographs. Evidence strength summarizes model directness, mechanistic tests and timing relevance. Model categories: A: In vivo myocardial ischemia/reperfusion (I/R) in an intact animal; B: Isolated-heart I/R without intact systemic context; C: Cellular hypoxia/reoxygenation (R) or oxygen-glucose deprivation/R surrogates; D: Permanent myocardial ischemia/myocardial infarction (MI) without reperfusion; E: Other injury models. A/B directly model cardiac I/R; C models cellular components; D/E provide indirect evidence. Categories are not overall evidence-quality ranks. Assoc: No targeted causal test; named perturbations support the tested pathway, not necessarily fission mediation; comparators do not establish necessity. Pre: Before injury (post-onset initiation untested); Isch: During ischemia; Rep: At/after reperfusion onset. Acute-treatment relevance depends on start time, model, route and exposure; Pre + Rep does not establish Rep-only efficacy. Post-MI: After non-reperfused MI (post-infarction recovery). Cellular timing does not establish clinical windows; timing relevance does not establish clinical efficacy. Sample sizes are endpoint-specific and per group unless independent experiments are specified; cellular n retains the reported replication unit. Only explicitly reported outcome values are shown; graph estimates are excluded. ≈ denotes an approximation stated by the source authors; dispersion is retained as reported. Tongmai Yangxin Pill timing is inconsistently reported in Yu et al[84], precluding a firm timing-relevance assessment. R: Randomization; B: Reported blinding stage; yes: Reported; NR: Not reported; GRS: Ginsenoside Rb1-ruscogenin-schisandrin combination; LAD: Left anterior descending coronary artery; I/R: Ischemia/reperfusion; H/R: Hypoxia/reoxygenation; Drp1: Dynamin-related protein 1; AMPK: AMP-activated protein kinase; NRVMs: Neonatal rat ventricular myocytes; MED: Minimum effective dose; MEC: Minimum effective concentration; EF: Ejection fraction; Bcl-2/Bax: B-cell lymphoma 2 to Bcl-2-associated X protein; LDH: Lactate dehydrogenase; AMPK: AMP-activated protein kinase; PBM: Proprietary botanical medicine; MI: Myocardial infarction; LAD: Left anterior descending coronary artery; SMI: Shenmai Injection; FS: Fractional shortening; LC3-II: Light chain 3-II; ΔΨm: Mitochondrial membrane potential; Fis1: Mitochondrial fission 1 protein; mPTP: Mitochondrial permeability transition pore; CRF: Chronic renal failure; GXN: Guanxinning Injection; MFN1: Mitofusin 1; OPA1: Optic atrophy 1; cTnT: Cardiac troponin T; CK-MB: Creatine kinase-MB; CMECs: Cardiac microvascular endothelial cells; OGD/R: Oxygen-glucose deprivation/reoxygenation; SSNX: Shuangshen Ningxin Formula; NR4A1: Nuclear receptor subfamily 4 group A member 1; Mff: Mitochondrial fission factor; HK II: Hexokinase II; Cyt c: Cytochrome c; THSWD: Taohong Siwu Decoction; LVESV: Left ventricular end-systolic volume; ROS: Reactive oxygen species; Akt: Protein kinase B; QSYQ: Qishen Yiqi Dripping Pill; LVEF: Left ventricular ejection fraction; PGC-1α: Peroxisome proliferator-activated receptor-γ coactivator 1α; TFAM: Mitochondrial transcription factor A; DQ: Danqi Soft Capsule; TMYX: Tongmai Yangxin Pill; cTnI: Cardiac troponin I; ERα: Estrogen receptor α; PC6: Pericardium 6, Neiguan; ST36: Stomach 36, Zusanli; CV4: Conception vessel 4, Guanyuan; EA: Electroacupuncture; SOD: Superoxide dismutase; MDA: Malondialdehyde; NRF2: Nuclear factor erythroid 2-related factor 2.

MITOCHONDRIAL FUSION AND FISSION IN CARDIOMYOCYTES: PHYSIOLOGICAL ROLES AND MOLECULAR MECHANISMS

Mitochondrial morphology in cardiomyocytes is maintained through the coordinated activity of fusion and fission. Mitochondrial fusion involves the merger of the outer and inner membranes of adjacent mitochondria, whereas mitochondrial fission divides a mitochondrion into separate daughter organelles[10]. These processes are not intrinsically protective or detrimental. Instead, their physiological significance depends on their coordination with cellular energy demand, mitochondrial quality control and organelle turnover.

Physiological roles of mitochondrial fusion and fission

Because cardiomyocytes have a high and continuous demand for ATP, they depend on the structural and functional integrity of their mitochondrial population. Mitochondrial fusion facilitates the exchange of matrix contents, membrane components and mitochondrial genetic material between adjacent mitochondria. By allowing partially impaired mitochondria to share functional components with healthier organelles, fusion supports functional complementation, respiratory efficiency and maintenance of mitochondrial network integrity. Fusion also contributes to the preservation of cristae structure and membrane potential, thereby sustaining oxidative phosphorylation under physiological stress.

Mitochondrial fission plays a complementary role. It facilitates the distribution and repositioning of mitochondria, permits adaptation of mitochondrial morphology to changing metabolic requirements and segregates severely damaged mitochondrial regions from the remaining network. These damaged components can then be recognized and removed through mitochondrial quality-control pathways[11].

The functional state of the mitochondrial population depends on the relative activity of these two processes. Fusion enables content exchange and functional compensation, whereas fission supports segregation, redistribution and quality control. Together, these processes help preserve mitochondrial morphology, membrane potential, respiratory-complex activity and energy production in cardiomyocytes[12,13].

Mitochondrial fusion: Tethering and coordinated outer-membrane and inner-membrane fusion

Mitochondrial fusion is a multistep process involving initial mitochondrial tethering and docking, followed by fusion of the OMM and inner mitochondrial membrane (IMM). Fusion begins when two mitochondria are brought into close proximity. MFN1 and MFN2, located in the OMM, are the principal guanosine triphosphatases responsible for mitochondrial tethering and outer-membrane fusion[14]. Mitofusin molecules on apposed mitochondria can form trans assemblies that stabilize intermitochondrial contact[15]. Guanosine triphosphate (GTP) binding and hydrolysis, together with mitofusin oligomerization and associated conformational rearrangements, promote close membrane apposition and outer-membrane merger[16,17]. In vitro studies using isolated yeast mitochondria have visualized mitofusin-dependent docking structures and local fusion at expanded contact regions, although it remains unclear whether mammalian mitochondria use an identical architecture[16]. OMM fusion generally precedes the subsequent fusion of the inner membranes[18].

OPA1, a dynamin-related GTPase anchored in the IMM and exposed predominantly to the intermembrane space, is the principal mediator of inner-membrane fusion and also contributes to cristae organization and mitochondrial function[19]. OPA1 exists as membrane-anchored long forms and soluble short forms generated by proteolytic processing[20,21]. An appropriate balance between these forms supports efficient inner-membrane fusion. Long OPA1 provides a membrane-anchored, fusion-competent form, whereas stoichiometric amounts of short OPA1 can cooperate with long OPA1 to facilitate efficient completion of membrane fusion; excessive processing and loss of long OPA1 impair fusion competence[20,22]. Reconstitution studies have shown that membrane-anchored long-form OPA1 (L-OPA1) on one membrane can cooperate with cardiolipin in the opposing membrane to support inner-membrane tethering and fusion[23]. Structural studies of the yeast OPA1 orthologue Mgm1 have identified higher-order assemblies compatible with membrane remodeling[20], whereas more recent structural work indicates that human OPA1 forms membrane-associated oligomeric assemblies capable of remodeling cardiolipin-containing membranes[24]. Following OMM fusion, OPA1-dependent IMM fusion establishes continuity between the mitochondrial matrices, enabling exchange of mitochondrial contents and formation of a larger, continuous mitochondrial structure[18,25].

Mitochondrial fission: Drp1 recruitment, membrane constriction and scission

Mitochondrial fission involves selection of a division site, local membrane remodeling, recruitment and assembly of the fission machinery, and final membrane scission. Fission frequently begins at contact sites between mitochondria and the endoplasmic or sarcoplasmic reticulum, where local remodeling helps define and partially constrict the future division site[26-28].

Drp1 is predominantly cytosolic under basal conditions. In response to fission-promoting signals, Drp1 is recruited to the OMM through interactions with several membrane-associated receptor or adaptor proteins, including Mff, Fis1, MiD49 and MiD51. These proteins contribute to Drp1 recruitment and regulation at the OMM in distinct but partly overlapping ways[28].

After recruitment, Drp1 oligomerizes around the mitochondrial tubule and forms ring-like or spiral-like structures at the prospective fission site. GTP-dependent conformational changes within the Drp1 complex generate mechanical force that progressively constricts the mitochondrial membranes and promotes division of the organelle[29]. Drp1-dependent mitochondrial fission is regulated at multiple levels, including post-translational modifications, cytosol-to-mitochondria translocation, higher-order assembly at fission sites, and interactions with OMM-associated receptor or adaptor proteins[30].

Among the known Drp1-associated outer-membrane proteins, Mff has a well-established role in mammalian mitochondrial fission. Mff depletion markedly impairs mitochondrial recruitment of Drp1, whereas Mff overexpression promotes Drp1 recruitment and mitochondrial fragmentation[31,32]. MiD49 and MiD51 are vertebrate OMM adaptors that can recruit Drp1 independently of Mff and Fis1 and influence its organization at prospective fission sites[7,33]. However, Drp1 recruitment by MiD49/51 does not necessarily indicate productive fission, because Mff and MiD49/51 can preferentially associate with different Drp1 assembly states[34]. Fis1 does not appear to function as a universal Drp1 receptor in mammalian cells, and its contribution may be more selective. Live-cell imaging studies have associated Fis1-enriched peripheral fission with the segregation of dysfunctional mitochondrial material, whereas Mff-enriched midzone fission has been linked more closely to mitochondrial proliferation or biogenesis[35]. These distinctions indicate that Mff, Fis1, MiD49 and MiD51 should not be treated as functionally interchangeable components of the mammalian fission machinery. The principal molecular events involved in mitochondrial fusion and fission are summarized in Figure 1.

Figure 1 Molecular machinery of mitochondrial fusion and fission. Mitochondrial fusion involves mitofusin 1 (MFN1) and MFN2, the long forms of optic atrophy 1 (L-OPA1) and short forms of optic atrophy 1 (S-OPA1), and cardiolipin (CL). MFN1 and MFN2 mediate outer mitochondrial membrane (OMM) tethering and fusion through trans interactions, oligomeric rearrangement, and guanosine triphosphate hydrolysis. At the inner mitochondrial membrane, membrane-anchored L-OPA1 interacts with CL on the opposing membrane to promote membrane tethering and fusion. S-OPA1 can cooperate with L-OPA1 in OPA1-dependent membrane remodeling, although the precise functional division between the two forms remains incompletely defined. Mitochondrial fission involves the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), dynamin-related protein 1 (Drp1), and OMM-associated receptor or adaptor proteins, including mitochondrial fission factor, mitochondrial fission 1 protein, and mitochondrial dynamics proteins of 49 kDa and mitochondrial dynamics proteins of 51 kDa (MiD49 and MiD51). ER/SR-mitochondria contact sites mark and partially constrict prospective division sites. Drp1 is subsequently recruited from the cytosol to the OMM, where it assembles into oligomeric rings or helices. Guanosine triphosphate hydrolysis drives conformational changes in the Drp1 oligomer, promoting further membrane constriction and mitochondrial scission. MFN1/2: Mitofusin 1/2; L-OPA1: Long forms of optic atrophy 1; S-OPA1: Short forms of optic atrophy 1; CL: Cardiolipin; ER: Endoplasmic reticulum; SR: Sarcoplasmic reticulum; Drp1: Dynamin-related protein 1.

PATHOPHYSIOLOGICAL RELEVANCE OF EXCESSIVE MITOCHONDRIAL FISSION IN MI/RI

The coordination between mitochondrial fusion and fission described above is markedly disrupted during MI/RI. Ischemia and subsequent reperfusion expose cardiomyocytes to abrupt metabolic, ionic and oxidative disturbances that destabilize mitochondrial membranes and shift the mitochondrial population toward a fragmented phenotype. This transition is driven predominantly by Drp1-centered excessive fission, whereas insufficient MFN1/2-mediated and OPA1-mediated fusion limits the reassembly and functional recovery of the fragmented mitochondrial network. The resulting fission-dominant imbalance contributes not only to cardiomyocyte bioenergetic failure and death but also to impaired coronary microvascular recovery.

Reperfusion-associated stress and mitochondrial destabilization

During myocardial ischemia, interrupted oxygen and substrate delivery suppresses mitochondrial oxidative phosphorylation and reduces ATP production. The resulting energy deficit compromises ATP-dependent ion transport, promotes intracellular Ca²⁺ accumulation and disturbs cellular acid-base homeostasis. Although these changes originate during ischemia, they also render mitochondria particularly vulnerable to the abrupt restoration of oxygen and metabolic activity at the onset of reperfusion[36].

Rapid reoxygenation promotes excessive ROS generation, while intracellular and mitochondrial Ca²⁺ overload persists or worsens. Together, these disturbances increase mitochondrial membrane permeability and facilitate opening of the mPTP. Sustained mPTP opening causes mitochondrial swelling, impairs electron transport chain activity, destabilizes the OMM and dissipates the ΔΨm[37,38]. Loss of ΔΨm further compromises ATP synthesis and facilitates the release of pro-apoptotic factors, including cytochrome c, from mitochondria into the cytosol. ROS accumulation, Ca²⁺ overload, mPTP opening and ΔΨm dissipation constitute a mutually reinforcing process of mitochondrial destabilization. Increased intracellular Ca²⁺ can promote mitochondrial division in ventricular cardiomyocytes[39], whereas ROS-dependent signaling can enhance Drp1 activation and mitochondrial recruitment[40]. Conversely, excessive mitochondrial fragmentation may aggravate respiratory dysfunction, ROS generation and membrane-potential loss, thereby further destabilizing the mitochondrial population.

Drp1-centered fission activation and pathological mitochondrial fragmentation

Drp1-centered activation is a principal molecular mechanism linking reperfusion-associated stress to pathological mitochondrial fragmentation. During MI/RI, however, abnormal post-translational regulation and enhanced mitochondrial translocation of Drp1 promote excessive mitochondrial fission[41].

Phosphorylation at specific Drp1 residues provides an important regulatory link between reperfusion-associated stress and fission activation. In HL-1 cardiomyocytes subjected to anoxia/reoxygenation, increased ROS production was accompanied by enhanced Drp1 phosphorylation at Ser616, whereas phosphorylation at the inhibitory Ser637 site was reduced. ROS scavenging suppressed the increase in Ser616 phosphorylation but did not prevent the reduction in Ser637 phosphorylation. Protein kinase C delta knockdown and pharmacological cyclin-dependent kinase inhibition attenuated Ser616 phosphorylation, mitochondrial fragmentation and cardiomyocyte injury, whereas calcineurin inhibition preserved Ser637 phosphorylation and reduced mitochondrial fragmentation[40]. These findings suggest that anoxia/reoxygenation-induced fission may involve both enhanced activating phosphorylation and reduced inhibitory phosphorylation of Drp1.

More recent evidence supports Ser637-dependent control of Drp1 mitochondrial recruitment. In H/R-treated HL-1 cells, Opa-interacting protein 5 antisense ribonucleic acid 1 (Oip5-as1) bound A-kinase anchoring protein 1 and calcineurin and promoted their association, reducing calcineurin activity and preserving Drp1 Ser637 phosphorylation. Oip5-as1 overexpression limited Drp1 mitochondrial translocation and mitochondrial fragmentation and attenuated injury in H/R-treated cells and mouse I/R models[41]. These findings provide a more direct mechanistic link between Drp1 phosphorylation, mitochondrial localization, fragmentation and myocardial injury.

Drp1 recruitment also depends on receptor and adaptor proteins located at the OMM. Recent evidence has identified a nuclear receptor subfamily 4 group A member 1-Mff (NR4A1-Mff) pathway in cardiac I/R injury, in which increased NR4A1 signaling enhanced Mff-associated mitochondrial fission and aggravated myocardial injury[42]. These findings broaden the mechanistic picture beyond changes in total Drp1 abundance and highlight the contribution of outer-membrane fission machinery to cardiac I/R injury. Experimental inhibition of Drp1-associated fission has been shown to reduce mitochondrial fragmentation, delay mPTP opening and limit myocardial infarct size[6]. Drp1 inhibition initiated after ischemia but before reperfusion also improved post-ischemic left ventricular function, supporting a functional relationship between excessive fission and reperfusion-associated cardiac dysfunction[5].

Together, these observations indicate that mitochondrial fragmentation is not merely a passive morphological marker of injured cardiomyocytes. Drp1-centered excessive fission actively contributes to bioenergetic failure, membrane instability and cell-death signaling. Nevertheless, fragmentation cannot be attributed to increased fission alone, because the persistence of a fragmented mitochondrial population also depends on whether fusion can restore network continuity.

Impaired mitochondrial fusion and failure of network restoration

During MI/RI, insufficient MFN1/2-mediated and OPA1-mediated fusion reduces the capacity of fragmented mitochondria to reconnect and recover after acute reperfusion stress. Evidence from genetic and pharmacological studies supports the importance of fusion capacity in determining myocardial susceptibility to I/R injury. Opa1 haploinsufficiency increases cardiac sensitivity to I/R and is associated with impaired mitochondrial function and altered mitochondrial Ca²⁺ handling[43]. Conversely, in prediabetic rats subjected to cardiac I/R, administration of the mitochondrial fusion promoter M1 during ischemia or at the onset of reperfusion reduced infarct size and apoptosis-related changes and improved left ventricular function[44]. Overexpression of MFN1 or MFN2 has also been reported to reduce susceptibility to mPTP opening and cell death after simulated I/R[6]. However, these findings should not be interpreted as evidence that MFN1/2 and OPA1 are uniformly downregulated in every MI/RI model. The expression, processing and function of fusion-related proteins may vary according to the duration of ischemia, reperfusion interval, model system and myocardial cell type. A more cautious interpretation is that MI/RI can compromise MFN1/2-dependent and OPA1-dependent fusion capacity, thereby reducing the ability of mitochondria to restore structural continuity and functional complementation.

Failure to restore the mitochondrial network has consequences beyond morphology. Restricted fusion limits the exchange of respiratory proteins, metabolites and mitochondrial genetic material between damaged and relatively preserved organelles. It may also impair maintenance of cristae architecture, membrane potential and oxidative phosphorylation. Consequently, fragmented mitochondria remain functionally isolated and are less able to compensate for local injury. Impaired fusion therefore acts as an amplifying and sustaining mechanism that complements excessive fission rather than as an unrelated pathological process. Drp1-centered activation increases the production of fragmented mitochondria, whereas insufficient MFN1/2-mediated and OPA1-mediated fusion prevents these fragments from reconnecting and recovering. Together, these changes establish a fission-dominant fusion-fission imbalance in which excessive division initiates and expands mitochondrial fragmentation, while impaired fusion prolongs network disruption.

This distinction also matters when interpreting cardioprotective interventions. Suppression of Drp1-associated fission limits the formation of additional mitochondrial fragments, whereas restoration of MFN1/2-mediated or OPA1-mediated fusion may facilitate network repair. Although both effects may contribute to mitochondrial preservation, suppression of excessive fission and restoration of fusion capacity represent complementary but mechanistically distinct routes to network recovery.

Consequences for cardiomyocyte survival and coronary microvascular function

The pathological significance of fission-dominant fusion-fission imbalance ultimately lies in its effects on cardiomyocyte survival and post-ischemic recovery. Excessive Drp1-associated fission increases mitochondrial fragmentation and disrupts maintenance of ΔΨm, oxidative phosphorylation and Ca²⁺ homeostasis. These changes reduce ATP availability, facilitate mPTP opening and mitochondrial permeabilization, and promote the release of pro-apoptotic factors, thereby increasing cardiomyocyte death and myocardial injury[38,40,45]. Experimental inhibition of Drp1 recruitment or activity reduces mitochondrial fragmentation, cardiomyocyte death and infarct size and improves post-ischemic ventricular function, supporting a functional contribution of excessive fission rather than a purely morphological association[5,46]. Conversely, insufficient MFN1/2-dependent or OPA1-dependent fusion may restrict functional complementation and network recovery. Increased susceptibility of Opa1-haploinsufficient hearts to I/R further supports the importance of preserved fusion-related capacity, although the contribution of OPA1 cannot be separated completely from its additional roles in cristae organization and mitochondrial Ca²⁺ handling[43].

Mitochondrial disruption may also affect the coronary microvasculature. Endothelial cells require coordinated mitochondrial function to support migration, angiogenic responses and nitric oxide-dependent vascular regulation. Experimental depletion of MFN1 or MFN2 impairs endothelial-cell migration and tube formation, indicating that mitofusin-dependent mitochondrial organization contributes to normal endothelial repair and angiogenic function[47]. More broadly, endothelial mitochondrial dysfunction has been associated with impaired endothelial nitric oxide synthase activation, reduced nitric oxide availability and defective endothelium-dependent vasomotor responses[48]. These findings do not establish reduced fusion as the sole cause of coronary microvascular dysfunction during MI/RI; however, they indicate that disruption of mitochondrial structure and function can compromise endothelial adaptive capacity.

During cardiac microvascular I/R injury, oxidative stress, mitochondrial membrane damage and impaired vasodilatory signaling contribute to endothelial dysfunction, capillary obstruction and incomplete microvascular reperfusion despite restoration of epicardial coronary flow[49]. Persistent mitochondrial fragmentation may aggravate these processes by amplifying ROS production and limiting mitochondrial functional recovery, whereas impaired endothelial migration and angiogenic capacity may further delay vascular repair. Thus, fission-dominant mitochondrial disruption may influence MI/RI at two interconnected levels: By promoting energetic failure and death in cardiomyocytes and by impairing endothelial and coronary microvascular function. This provides the rationale for examining whether plant-derived natural products, multi-component botanical preparations and electroacupuncture can restrain pathological fission, preserve fusion-related capacity and protect both myocardial and microvascular compartments during MI/RI.

PLANT-DERIVED INTERVENTIONS AND ELECTROACUPUNCTURE: POTENTIAL MODULATION OF FISSION-DOMINANT MITOCHONDRIAL FUSION-FISSION IMBALANCE IN MI/RI AND RELATED EXPERIMENTAL MODELS

Some interventions are associated with changes in core components of the fusion-fission machinery, including Drp1, Fis1, MFN1/2 and OPA1, whereas others primarily modulate upstream pathways linked to mitochondrial morphology, bioenergetics, oxidative stress and cell-death signaling. To facilitate comparison across these evidence levels, Tables 1 and 2 summarize the experimental models, intervention regimens, molecular targets and principal outcomes of the included studies. Botanical nomenclature, compound identity and chemical classification were standardized using authoritative botanical and chemical databases, including Plants of the World Online, PubChem and ClassyFire, and pharmacopoeial names were checked against relevant pharmacopoeial sources where applicable. Assessments of botanical-intervention reporting quality, informed by the Consensus statement on Phytochemical Characterisation of Medicinal Plant extracts (ConPhyMP), are provided in Supplementary Tables 1 and 2.

Plant-derived natural products potentially modulating excessive fission and impaired fusion

Chemically defined plant-derived compounds permit more precise characterization of exposure and molecular effects than multi-component preparations. The available studies associate these compounds with three partly overlapping response patterns: Changes in Drp1-associated fission signaling, changes in MFN1/2-related or OPA1-related fusion markers, and modulation of upstream pathways linked to mitochondrial structure and function.

Triterpenoid saponins: Ginsenosides are pharmacologically active triterpenoid saponins derived from Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma), with numerous structurally distinct metabolites identified to date[50]. Their biological activities have been investigated in cardiovascular and other ischemia-related disorders[51,52]. Ginsenoside Rg1 has primarily been associated with fusion-related mitochondrial remodeling, although both the direction of MFN2 regulation and the directness of the supporting measurements vary across experimental models.

In an in vitro cardiomyocyte H/R model, Rg1 increased MFN2 protein expression and dose-dependently upregulated MFN2 mRNA, and also modulated glutamate dehydrogenase activity and intracellular glutamate levels. MFN2 silencing abolished the cytoprotective effect of Rg1, supporting a functional role for MFN2 in Rg1-mediated protection against H/R-induced mitochondrial dysfunction and cardiomyocyte injury[53]. By contrast, another study in H/R-treated HL-1 cells found that Rg1 increased Notch1 and MFN1 expression but reduced the H/R-associated elevation of MFN2. Notch1 silencing attenuated these effects, suggesting that Notch1-dependent remodeling contributed to the regulation of fusion-related proteins[54]. The opposing directions of MFN2 regulation in these studies do not support describing Rg1 as a simple MFN2 activator. Differences in cell type, H/R duration, intervention concentration, pretreatment schedule and Notch1-dependent responses may have contributed to the discrepancy. Rg1 is therefore more appropriately regarded as a context-dependent modulator of fusion-related responses rather than as an intervention that uniformly increases MFN2 or promotes mitochondrial fusion.

Ginsenoside Rg5 provides direct cellular evidence of mitochondrial fission inhibition. Mitochondrial retention of hexokinase II (HK II) is associated with resistance to ischemic injury, whereas HK II dissociation correlates with cytochrome c release, ROS generation and infarct size during reperfusion[38]. In palmitic acid-sensitized neonatal rat ventricular myocytes subjected to H/R, Rg5 promoted mitochondrial translocation of protein kinase B (Akt), reduced mitochondrial recruitment of Drp1 and preserved mitochondrial HK II binding, thereby reducing mitochondrial fragmentation and cellular injury[55]. These cellular findings support a mechanism centered on the reciprocal regulation of mitochondrial Drp1 recruitment and HK II retention. However, the accompanying mouse experiment used isoproterenol-induced injury rather than myocardial I/R, and whether Rg5 also restores MFN1/2-dependent or OPA1-dependent fusion has not been established.

Ginsenoside Rc has been proposed to preserve mitochondrial function through the sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway. Rc directly interacted with and activated SIRT1, promoted PGC-1α deacetylation and improved cellular energy metabolism under I/R-related conditions. The source study also reported increased nuclear respiratory factors 1 and 2 in H/R-treated cells, but neither factor was independently manipulated[56]. In a separate experimental context, activation of SIRT1/PGC-1α was associated with increased MFN1 and MFN2 and reduced Drp1 and Fis1 expression[57]. These findings suggest a plausible link between Rc, metabolic regulation and fusion-fission homeostasis. Nevertheless, direct evidence that Rc regulates Drp1, MFN1/2 or OPA1 in direct in vivo or ex vivo models of myocardial I/R remains insufficient. Its relevance to fusion-fission regulation is therefore mechanistically plausible but remains indirect.

The astragaloside IV derivative LS-102 provides more direct evidence for site-specific regulation of Drp1. In cellular and animal I/R models, LS-102 activated phosphoinositide 3-kinase (PI3K)/Akt signaling, reduced Drp1 phosphorylation at Ser616 and increased phosphorylation at Ser637, thereby limiting Drp1-dependent mitochondrial fission, oxidative stress and cardiomyocyte apoptosis. The study further identified Drp1 Ser616 as a potential phosphorylation site regulated by glycogen synthase kinase-3β in cardiomyocyte I/R injury[58]. Thus, LS-102 may alleviate experimental MI/RI by shifting Drp1 phosphorylation away from a fission-promoting state.

Notoginsenoside R1, a protopanaxatriol-type saponin from Panax notoginseng (Burkill) F.H. Chen (Araliaceae; Notoginseng Radix et Rhizoma), has been associated with broader preservation of mitochondrial regulatory processes. In H/R-treated H9c2 cells, notoginsenoside R1 increased PGC-1α, nuclear respiratory factor 1 (NRF1), nuclear factor erythroid 2-related factor 2 (NRF2), and the fusion-related proteins OPA1, MFN1 and MFN2, while reducing Drp1 and Fis1. Changes in phosphatase and tensin homolog-induced kinase 1 (PINK1), parkin and B-cell lymphoma 2/adenovirus early region 1B 19-kDa-interacting protein 3 were also reported, indicating concurrent modulation of mitochondrial quality-control pathways[59]. The source study grouped both factors with mitochondrial-biogenesis markers; however, NRF1 and NRF2 are distinct regulators, and neither was independently manipulated. These results indicate a shift in fusion-fission-related protein expression, although actual mitochondrial fusion and fission events were not directly assessed; moreover, the concurrent changes in biogenesis and mitophagy make it difficult to attribute the protective effect specifically to fusion-fission regulation.

Calenduloside E (CE) provides comparatively strong mechanistic evidence for OPA1 involvement. In rat hearts subjected to I/R, CE partially restored OPA1 and MFN2 expression and reduced Drp1 expression, whereas in H/R-treated H9c2 cardiomyocytes it increased OPA1 and MFN2 while reducing Drp1 and Fis1. These changes were accompanied by lower levels of cleaved caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase. OPA1 silencing substantially attenuated the effects of CE on fusion-related and fission-related proteins, mitochondrial function, cell viability and apoptotic markers. CE also increased AMP-activated protein kinase (AMPK) phosphorylation, and pharmacological inhibition of AMPK attenuated its effects on OPA1 expression, ΔΨm and mPTP opening[60]. These findings support an OPA1-dependent component of CE-mediated cardioprotection and implicate AMPK as a potential upstream regulator; the accompanying changes in MFN2, Drp1 and Fis1 also indicate broader modulation of fusion-fission-related markers.

Collectively, the saponin studies point to several distinct levels of intervention. LS-102 and Rg5 provide direct evidence involving reduced mitochondrial fragmentation and Drp1 recruitment, CE supports an OPA1-dependent component of mitochondrial protection, and Rg1 shows context-dependent fusion-related responses. The evidence for ginsenoside Rc and notoginsenoside R1 is broader but less specific because their effects extend to energy metabolism, mitochondrial biogenesis and quality-control pathways.

Phenolic and phenylethanoid glycosides: Gastrodin is a major phenolic glycoside derived from Gastrodia elata Blume (Orchidaceae; Gastrodiae Rhizoma) and has antioxidant, anti-inflammatory and cytoprotective activities[61]. In cardiomyocytes exposed to hydrogen peroxide (H₂O₂), gastrodin promoted nuclear translocation of NRF2, increased MFN2 and OPA1 and reduced Fis1, thereby preserving mitochondrial structure and function and reducing oxidative injury[62]. The same study used an OGD/R-like protocol only for preliminary cell-viability and ROS experiments, whereas the principal fusion-fission-related and NRF2 mechanistic findings were obtained in the H₂O₂ model. Because oxidative stress is an important component of MI/RI[63], these findings provide supportive mechanistic evidence for potential gastrodin-mediated regulation of fusion and fission. Nevertheless, the core mechanistic evidence derives from an oxidative-stress model rather than direct in vivo or ex vivo myocardial I/R and should therefore be interpreted as supportive rather than direct evidence of efficacy in MI/RI.

Salidroside, a glycoside derived from Rhodiola rosea L (Crassulaceae; Rhodiolae roseae Rhizoma et Radix), was evaluated in a rat myocardial I/R model and in H9c2 cells subjected to H/R, an in vitro surrogate of MI/RI. Salidroside enhanced AMPK phosphorylation, alleviated endoplasmic reticulum stress and apoptosis, improved mitochondrial morphology and ATP production, restored MFN2 and OPA1 expression, and reduced Fis1 expression and Drp1 phosphorylation at Ser616 in I/R hearts. Compound C attenuated these effects in vivo and in vitro, whereas AMPK silencing similarly weakened the effects of salidroside on mitochondrial fragmentation, p-Drp1 Ser616, endoplasmic reticulum stress and apoptosis in H9c2 cells[64]. These findings implicate AMPK in salidroside-mediated regulation of mitochondrial fusion-fission-related changes, although they do not establish suppression of fission as the sole or necessary mediator of cardioprotection.

Flavonoid glycosides, isoquinoline alkaloids, and anthracenecarboxylic acids: Vitexin is a flavonoid glycoside found in Crataegus pinnatifida Bunge (Rosaceae; Crataegi Fructus) and has been investigated for antioxidant, anti-inflammatory and ischemia-related protective effects[65,66]. In isolated rat hearts subjected to I/R and H9c2 cells exposed to H/R, vitexin reduced exchange protein directly activated by cyclic adenosine monophosphate 1 and Ras-related protein 1 signaling, increased MFN2 and reduced Drp1 expression. These expression changes were consistent with reduced fission-related signaling and were accompanied by reduced oxidative stress and apoptosis[67]. A second study found that vitexin increased MFN2, reduced mitochondrial recruitment of Drp1, preserved outer-membrane and cristae structure and maintained ΔΨm during MI/RI[68]. Taken together, these studies support inhibition of Drp1-associated fission by vitexin, accompanied by increased MFN2 expression and preservation of mitochondrial structure.

Berberine, a major isoquinoline alkaloid found in Coptis chinensis Franch (Ranunculaceae; Coptidis Rhizoma), has been investigated in metabolic, gastrointestinal and cardiovascular disorders[69,70]. In a rat model of acute isoproterenol-induced β-adrenergic overactivation and in cobalt chloride-treated H9c2 cells, berberine pretreatment activated Akt and liver kinase B1/AMPK signaling, increased myocardial ATP levels, altered Drp1 phosphorylation and increased autophagy-related protein expression[71]. However, these models do not reproduce coronary ischemia followed by reperfusion, and the functional interpretation of Drp1 phosphorylation requires identification of the specific residue involved. These findings therefore do not establish that berberine suppresses pathological Drp1-dependent fission during MI/RI, but they provide indirect evidence that it may influence Drp1-associated mitochondrial regulation under β-adrenergic and hypoxic stress.

Rhein, an anthraquinone derivative found in Rheum palmatum L (Polygonaceae; Rhei Radix et Rhizoma), has been reported to have antioxidant and anti-inflammatory activities[72]. In a rat acute myocardial infarction model induced by left anterior descending coronary artery (LAD) ligation and in hypoxic H9c2 cells, rhein pretreatment increased OPA1 expression and reduced Drp1 expression. It also increased the mitophagy-related markers PINK1, Parkin and Beclin 1, the microtubule-associated protein 1 light chain 3-II (LC3-II)/LC3-I ratio and autolysosome formation; these changes were accompanied by reduced myocardial injury and cardiomyocyte apoptosis[73]. These results are consistent with reduced mitochondrial fission and enhanced mitophagy-related responses. However, increased OPA1 expression alone does not demonstrate restoration of mitochondrial fusion, and the dependence of rhein-mediated protection on the Drp1/PINK1/Parkin pathway was not confirmed through target-specific loss-of-function or rescue experiments. Because the study examined myocardial ischemia and cellular hypoxia without a clearly defined reperfusion phase, it provides supportive evidence from ischemic injury models but only indirect evidence for modulation of fission-dominant fusion-fission imbalance in MI/RI. Thus, mechanistic directness ranges from target manipulation and measurements of mitochondrial morphology or Drp1 recruitment to broader mitochondrial protection accompanied only by fusion-fission-related expression changes.

Multi-component botanical preparations potentially modulating mitochondrial fragmentation and network disruption

Multi-component botanical preparations differ from chemically defined compounds because their biological effects arise from combinations of constituents acting across multiple molecular pathways. Reported outcomes commonly include reduced fission-related signaling, increased fusion-related protein expression, preservation of mitochondrial morphology and attenuation of oxidative stress and apoptosis. However, the complexity of these preparations also makes attribution to a single active constituent or molecular target more difficult.

Shenmai Injection is derived from extracts of Panax ginseng C.A. Mey (Araliaceae; Ginseng Radix et Rhizoma) and Ophiopogon japonicus (Thunb) Ker Gawl (Asparagaceae; Ophiopogonis Radix).

In H/R-treated H9c2 cells, Shenmai Injection increased MFN1, MFN2 and OPA1 mRNA and reduced Drp1 and Fis1 mRNA. Alongside these molecular changes, LC3, Beclin 1, PINK1 and Parkin increased, indicating concurrent changes in fusion-control-related, fission-control-related and quality-control-related markers. These changes were accompanied by improved mitochondrial function and reduced cellular injury. Cardiac protection was also observed after permanent LAD ligation in rats, but this in vivo model did not include reperfusion[74]. Accordingly, the mechanistic evidence comes mainly from a cellular H/R surrogate, whereas the animal findings relate to permanent ischemic injury. Overall, these findings support a broad improvement in mitochondrial homeostasis; however, the active constituents responsible and the respective contributions of fusion-fission regulation and mitophagy to the protective effect remain unresolved. In the animal study, Shenmai Injection was administered after permanent LAD ligation rather than as pretreatment.

Guanxinning Injection contains extracts of Salvia miltiorrhiza Bunge (Lamiaceae; Salviae Miltiorrhizae Radix et Rhizoma) and Ligusticum chuanxiong Hort (Apiaceae; Chuanxiong Rhizoma). In rats with chronic renal failure complicated by myocardial I/R, both ischemic postconditioning alone and its combination with Guanxinning Injection reduced total Drp1 expression and increased MFN1 and OPA1 relative to the untreated comorbid group. The addition of Guanxinning Injection further increased MFN1 and OPA1 expression compared with ischemic postconditioning alone, whereas Fis1 remained unchanged. The combined intervention was also associated with preserved mitochondrial ultrastructure, reductions in myocardial injury markers and infarct size, and less histological damage[75]. These findings associate the addition of Guanxinning Injection with enhanced expression of fusion-related proteins and additional cardioprotection in a renal-comorbidity model. However, because no Guanxinning Injection-only group was included, its independent effects and its interaction with ischemic postconditioning could not be determined.

Danqi Soft Capsule contains Salvia miltiorrhiza Bunge and Panax notoginseng (Burkill) F.H. Chen. In rats with MI/RI, Danqi Soft Capsule reduced infarct size and cardiomyocyte apoptosis, improved mitochondrial morphology and decreased fragmentation. The intervention reduced Ca²⁺/calmodulin-dependent protein kinase II activation and Drp1 phosphorylation and was associated with preservation of ΔΨm, inhibition of mPTP opening and reduced ROS generation[76]. These findings link suppression of Drp1-associated fission with preservation of mitochondrial membrane function and inhibition of mitochondria-mediated apoptosis. Because transmission electron microscopy was used to evaluate mitochondrial morphology, the study provides structural as well as molecular evidence of reduced fragmentation.

Qishen Yiqi Dripping Pill contains multiple constituent groups, including astragalosides, tanshinones and ginsenosides, and has been reported to exert antioxidant, anti-inflammatory, anti-apoptotic and antiplatelet effects[77,78]. In a mouse model of post-MI cardiac dysfunction induced by permanent LAD ligation, Qishen Yiqi treatment reduced the p-Drp1 (Ser616)/Drp1 ratio and increased MFN2 expression and the L-OPA1/S-OPA1 ratio, particularly at the highest tested dose. It also increased the mitochondrial biogenesis-related proteins PGC-1α, mitochondrial transcription factor A (TFAM) and NRF1, improved mitochondrial ultrastructure, reduced mitochondrial ROS and attenuated ferroptosis-related biochemical and molecular changes. These effects were accompanied by improved cardiac function, and similar marker changes were observed in H9c2 cells exposed to OGD without reoxygenation[79]. These findings are consistent with reduced pro-fission signaling, preservation of fusion-related markers and enhanced mitochondrial biogenesis-related signaling. However, actual mitochondrial fission and fusion events were not directly measured, and the causal roles of Drp1, MFN2 or OPA1 were not tested. Because neither permanent coronary ligation nor OGD involved reperfusion, the study provides indirect evidence for MI/RI.

A three-component combination derived from Shengmai San, consisting of ginsenoside Rb1, ruscogenin and schisandrin, provides evidence for AMPK-dependent regulation of excessive fission. In a mouse myocardial I/R model and H/R-treated cardiomyocytes, the combination reduced Drp1 Ser616 phosphorylation, limited Drp1 mitochondrial recruitment and decreased mitochondrial fragmentation and apoptosis. In the cellular H/R system, the roles of AMPK activation and inhibition were evaluated; inhibition of AMPKα weakened these effects, supporting a functional relationship between AMPK activation and suppression of Drp1-centered fission[80]. This intervention therefore represents a relatively clear example of an upstream energy-sensing pathway converging on the fission machinery.

Taohong Siwu Decoction has been investigated for anti-inflammatory, antioxidant, antifibrotic and vascular effects[81]. In rats subjected to permanent LAD ligation, Taohong Siwu Decoction reduced Fis1 expression and mitochondrial ROS production, increased Akt phosphorylation, increased numbers of cluster of differentiation 31-positive microvessels and alpha-smooth muscle actin-positive vessels, and reduced collagen deposition and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells in the infarcted myocardium[82]. These findings suggest that suppression of fission-related signaling may accompany improvements in the ischemic myocardial microenvironment. However, because the experimental model did not include a clearly defined reperfusion phase, the evidence is more relevant to ischemic injury than to classical MI/RI.

Shuangshen Ningxin Formula was evaluated in rats subjected to myocardial I/R and in cardiac microvascular endothelial cells subjected to OGD/R. The formula reduced the transcription and protein expression of NR4A1, Mff and Drp1; these changes were accompanied by improved mitochondrial ultrastructure and function and reduced apoptosis[83]. Pharmacological inhibition of NR4A1 partially reproduced the cellular effects, supporting involvement of the NR4A1/Mff/Drp1 axis. However, mitochondrial fission events and Drp1 recruitment were not assessed directly.

Tongmai Yangxin Pill acts through a broader transcriptional and metabolic mechanism. In a rat myocardial I/R model and H/R-treated H9c2 cells, the formula activated the estrogen receptor α (ERα)/PGC-1α pathway, increased mitochondrial ATP production, reduced ROS and attenuated mitochondrial dysfunction and cell injury. In the cellular H/R model, silencing ERα weakened the protective response, supporting its role as an upstream mediator[84]. Unlike interventions that directly alter Drp1 phosphorylation or Mff expression, Tongmai Yangxin Pill may influence fusion-fission homeostasis indirectly through changes in mitochondrial bioenergetics and transcriptional regulation.

Across botanical preparations, recurrent changes in Drp1, Mff, Fis1, MFN1/2 or OPA1 were reported alongside upstream AMPK, PI3K/Akt, NR4A1/Mff/Drp1 or ERα/PGC-1α signaling; however, compositional differences, model heterogeneity and co-interventions limit direct comparison.

Electroacupuncture as a non-pharmacological intervention

Evidence for non-pharmacological modulation of mitochondrial fission in MI/RI remains limited. Electroacupuncture has been examined primarily as a pretreatment strategy rather than as an intervention initiated at reperfusion. AMPK can coordinate cellular energy and antioxidant responses, and AMPK-NRF2 signaling has been associated with reduced oxidative stress and Drp1-related fission under conditions of redox imbalance[85]. In a rat MI/RI model, electroacupuncture pretreatment increased myocardial AMPK and NRF2 protein expression and reduced Drp1, Mff and Fis1 expression, mitochondrial fragmentation and vacuolization. Compound C or ML385 attenuated these effects, providing pharmacological support for involvement of the AMPK-NRF2 axis, although phosphorylation of AMPK or NRF2 was not directly measured[86]. These findings suggest that electroacupuncture may limit fission-dominant mitochondrial disruption through an energy- and redox-sensitive pathway. However, the evidence derives from an animal study using a pretreatment design, and it remains uncertain whether similar effects would occur when electroacupuncture is administered during or after clinically initiated reperfusion.

Across the reviewed studies, reduced Drp1 recruitment or pro-fission phosphorylation, decreased Mff or Fis1 expression and improved mitochondrial morphology were recurrent patterns; fusion-related findings were less uniform, exemplified by opposite Rg1-associated MFN2 responses in two cellular H/R studies[53,54]. Taken together, these findings do not establish a universal mechanism. Direct in vivo myocardial I/R and ex vivo isolated-heart I/R were treated as distinct evidence categories; cellular H/R or OGD/R as surrogate evidence; and permanent ischemia or MI without reperfusion and other indirect injury models as indirect or supportive evidence. Figure 2 presents a proposed integrative summary rather than a single validated causal network.

Figure 2 Proposed pathways through which plant-derived interventions and electroacupuncture may influence fission-dominant mitochondrial disruption in myocardial ischemia/reperfusion injury and related experimental models. This integrative schematic summarizes reported pathways and molecular findings for chemically defined plant-derived compounds, multi-component botanical preparations and electroacupuncture in myocardial ischemia/reperfusion injury and related models. Reported upstream or associated routes include phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3β signaling and mitochondrial hexokinase II retention; liver kinase B1/AMP-activated protein kinase; sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) signaling associated with nuclear respiratory factors 1 and 2 and mitochondrial transcription factor A; estrogen receptor α/PGC-1α; AMPK-nuclear factor erythroid 2-related factor 2-associated antioxidant signaling; Notch1; exchange protein directly activated by cyclic adenosine monophosphate 1/Ras-related protein 1; nuclear receptor subfamily 4 group A member 1/mitochondrial fission factor (Mff); and Ca²⁺/calmodulin-dependent protein kinase II. Fusion-related findings include increased mitofusin 1 (MFN1) or optic atrophy 1 and context-dependent changes in MFN2. Fission-related findings include reduced Mff, mitochondrial fission 1 protein or dynamin-related protein 1 (Drp1), reduced Drp1 Ser616 phosphorylation and increased Drp1 Ser637 phosphorylation. Solid lines indicate within-study mechanistic support, dashed lines indicate associative relationships, and dotted blue lines indicate cross-study integrative or hypothesized links. This schematic does not represent a single experimentally validated causal network. Connections to grouped findings indicate pathway-level convergence rather than regulation of every marker; study-specific molecular associations, model directness, mechanistic causality and intervention timing are detailed in Tables 1 and 2. AMPK: AMP-activated protein kinase; OPA1: Optic atrophy 1; Epac1: Exchange protein directly activated by cyclic adenosine monophosphate 1; Rap1: Ras-related protein 1; Drp1: Dynamin-related protein 1; MFN1: Mitofusin 1; NFE2L2: Nuclear factor erythroid 2-related factor 2; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; GSK-3β: Glycogen synthase kinase 3β; LKB1: Liver kinase B1; HK II: Hexokinase II; CaMKII: Calmodulin-dependent protein kinase II; SIRT1: Sirtuin 1; PGC-1α: Peroxisome proliferator-activated receptor-γ coactivator 1α; NR4A1: Nuclear receptor subfamily 4 group A member 1; Mff: Mitochondrial fission factor; Fis1: Fission 1 protein.

INTEGRATED MECHANISTIC FRAMEWORK AND UNRESOLVED QUESTIONS

During myocardial I/R, redox, Ca²⁺, membrane-potential and kinase disturbances can converge on Drp1 recruitment through Mff, Fis1 and related outer-membrane adaptors. Sustained excessive fission is associated with fragmentation, impaired oxidative phosphorylation, ROS generation, mPTP opening and pro-death signaling; it may therefore amplify, rather than merely accompany, injury[87,88]. Experimental inhibition of Drp1 recruitment or activity has reduced fragmentation, infarct size and post-ischemic dysfunction in myocardial I/R models, supporting a contributory role[5,6].

The interventions reviewed above appear to act at several levels within this pathological network. Representative natural products, including ginsenosides, have been associated with preservation of mitochondrial integrity and regulation of MFN2- and Drp1-related processes, although their effects are not restricted to the fusion-fission machinery[89]. Other interventions have been linked to AMPK, PI3K/Akt, SIRT1/PGC-1α and related energy-responsive or stress-responsive signaling pathways[90-93]. These pathways should not be interpreted as components of a single linear cascade. Rather, they may constitute parallel upstream routes that influence Drp1 activation, mitochondrial bioenergetics, redox homeostasis, autophagy and cell survival to different degrees. Accordingly, concurrent changes in these pathways and mitochondrial morphology provide biological coherence but do not, by themselves, demonstrate that modulation of excessive fission is the necessary mediator of cardioprotection.

Several mechanistic uncertainties therefore remain. First, mitochondrial fragmentation may represent a cause, an amplifier or a consequence of injury depending on the severity and timing of ischemia and reperfusion. Measurements obtained at a single time point cannot distinguish an early adaptive response from persistent pathological fission. Second, total Drp1 abundance, site-specific phosphorylation, mitochondrial translocation, oligomerization and actual membrane scission are related but non-equivalent readouts. Although increased phosphorylation at Ser616 and decreased phosphorylation at Ser637 are frequently used as indicators of enhanced Drp1 activity, their functional interpretation depends on the upstream kinase, experimental context and accompanying evidence of Drp1 recruitment and mitochondrial division. Third, fission cannot be evaluated independently of fusion, mitophagy and mitochondrial biogenesis. Physiological Drp1-dependent fission contributes to the segregation and removal of damaged mitochondria, and sustained or complete Drp1 deficiency can impair mitochondrial quality control and cardiac function[94]. The relevant therapeutic objective is therefore the restriction of stress-induced excessive fission rather than indiscriminate suppression of Drp1 activity.

THERAPEUTIC IMPLICATIONS, EVIDENCE LIMITATIONS, AND FUTURE DIRECTIONS

The strength of this evidence is constrained at four independent levels. At the measurement level, many studies rely on total Drp1 expression, one phosphorylation site or static images of mitochondrial morphology without assessing Drp1 translocation, fission events, respiratory function and mitochondrial quality control in the same experimental system. At the causal level, associations among an intervention, reduced fragmentation and cardioprotection are often not tested using genetic loss-of-function and gain-of-function approaches or rescue experiments. Results obtained with mdivi-1 also require caution because this compound can inhibit mitochondrial complex I and alter ROS production independently of direct Drp1 inhibition[95]. At the translational level, substantial heterogeneity in cell type, animal species, ischemia duration, reperfusion interval, intervention timing and outcome definitions limits comparisons among studies. At the model level, this review distinguishes direct in vivo myocardial I/R from ex vivo isolated-heart I/R because only the former retains systemic physiological context. Cellular H/R or OGD/R studies provide surrogate evidence, while permanent ischemia or MI without reperfusion and other indirect injury models offer only indirect mechanistic support for MI/RI. A study can therefore be model-direct yet mechanistically correlative, or causally informative yet clinically mistimed; these dimensions should not be conflated. H9c2 cells and otherwise healthy young rodents are useful for initial screening but do not adequately reproduce adult cardiomyocyte architecture, coronary reperfusion or the comorbidities and concomitant medications commonly present in patients. Botanical extracts and compound formulations introduce additional uncertainty when their source material, chemical composition, pharmacologically relevant exposure and batch consistency are incompletely characterized. The narrative selection of representative studies may introduce selective citation, while publication bias may underrepresent negative or null findings and overstate the consistency of cardioprotection.

Future studies should address these limitations using an integrated rather than marker-centered design. Quantitative assessment of mitochondrial morphology should be combined with membrane potential, respiration, ATP production, ROS generation, Ca²⁺ handling and mPTP activity at defined stages of ischemia and reperfusion. Drp1 abundance and phosphorylation should be complemented by its subcellular localization, interaction with Mff or other relevant receptors, oligomerization and, where feasible, live-cell measurement of fission events. Genetic or selective pharmacological loss-of-function, gain-of-function and rescue experiments are needed to determine whether modulation of excessive fission is necessary and sufficient for the observed protection, and whether upstream pathways act directly on the fission machinery or indirectly by reducing the overall burden of mitochondrial stress. Parallel evaluation of MFN1/2, OPA1, mitophagy and mitochondrial biogenesis would further distinguish restoration of coordinated mitochondrial homeostasis from isolated changes in a single protein.

Experimental progression should follow established principles of rigor and translational relevance in myocardial I/R research, including randomization, blinding, prespecified outcomes, appropriate sample size estimation and standardized reporting of ischemia, reperfusion and treatment protocols[96,97]. Findings from established cardiac cell lines and small-rodent models should be validated in primary adult cardiomyocytes, human induced pluripotent stem cell-derived cardiac models, large animals and models incorporating aging, diabetes or other clinically relevant comorbidities.

Intervention timing represents an additional translational limitation. Pre-ischemic treatment, particularly repeated pretreatment, may inform planned ischemic events but leaves efficacy following post-onset initiation untested. Administration during ischemia or early reperfusion may better approximate acute-treatment opportunities, although relevance depends on the exact timing, route and achievable exposure. Regimens combining pretreatment with reperfusion-phase dosing do not establish efficacy from reperfusion-only initiation. Treatment after non-reperfused MI informs post-infarction injury and recovery rather than acute reperfusion protection. Future studies should evaluate feasible post-onset schedules and distinguish administration during ischemia, immediately before or at reperfusion, and during early or delayed reperfusion[96,97].

For defined natural compounds, pharmacokinetic exposure, target engagement, structure-activity relationships, safety and potential interactions with antiplatelet, anticoagulant and other cardiovascular therapies should accompany mechanistic experiments. For botanical extracts and formulations, authentication of starting material, chemical profiling, batch-to-batch consistency and quality-control markers should meet established phytochemical reporting standards such as the ConPhyMP recommendations[98]. Regulatory requirements, product availability, manufacturing consistency and treatment resources may vary across regions and should be considered when assessing feasibility[99]. Electroacupuncture protocols likewise require reproducible treatment parameters, appropriate sham controls and consideration of operator training and resource availability[100]. These interventions should be investigated only as potential adjuncts and should not replace or delay standard reperfusion therapy.

CONCLUSION

Excessive mitochondrial fission represents a plausible amplifying process linking reperfusion-associated redox and Ca²⁺ stress to mitochondrial dysfunction and cardiomyocyte injury. Evidence from cellular H/R or OGD/R, permanent ischemia or MI without reperfusion, and other indirect injury models supports mechanistic plausibility but should be interpreted separately from direct in vivo myocardial I/R and ex vivo isolated-heart I/R evidence, which are treated here as distinct evidence categories. Plant-derived interventions and electroacupuncture have produced broadly consistent changes in upstream stress signaling, the Drp1-centered fission machinery and downstream mitochondrial and cardiac outcomes. Nevertheless, the evidence remains predominantly preclinical, multidirectional and correlative and therefore does not establish excessive fission as the direct or principal target of most interventions. Progress will require temporally resolved assessment of mitochondrial structure and function, causal manipulation of the fission machinery, rigorous characterization of interventions and validation in clinically relevant models. Clinical efficacy and safety of these interventions in MI/RI have not been established, and they should not replace or delay guideline-directed reperfusion therapy[101].