MAMs-Mediated Calcium Homeostasis in the Pathophysiology of Cardiovascular Diseases: A Mini-Review

Abstract

Cardiovascular diseases (CVDs) remain a leading threat to human health, imposing substantial morbidity and a considerable clinical burden worldwide. Disrupted mitochondrial calcium ions (Ca²⁺) homeostasis is increasingly recognized as a shared pathophysiological feature of diverse CVDs. Mitochondria and the endoplasmic reticulum (ER) are key organelles involved in intracellular Ca²⁺ homeostasis and energy metabolism, and their physical and functional coupling occurs at mitochondria-associated ER membranes (MAMs). Through protein complexes such as IP3R–GRP75–VDAC1, VAPB–PTPIP51 and MFN1–MFN2, MAMs coordinate a finely tuned Ca²⁺-signalling network that is essential for maintaining mitochondrial Ca²⁺ homeostasis. This review provides a systematic overview of the structural organization of MAMs and the molecular mechanisms governing MAM-mediated Ca²⁺ transfer, with particular emphasis on how dysregulated Ca²⁺ signalling at MAMs contributes to myocardial ischaemia–reperfusion injury, atherosclerosis, heart failure (HF), pulmonary arterial hypertension and diabetic cardiomyopathy. Furthermore, on this basis, this review focuses on the IP3Rs‑GRP75‑VDAC1 complex, mitochondrial calcium uniporter (MCU)‑associated calcium‑transport pathways, the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis, as well as MAMs‑mediated signaling cascades, and systematically elaborates the latest research advances of therapeutic interventions including small‑molecule inhibitors, targeted peptides, calcium chelators, chemical chaperones and natural products. At present, available therapeutic evidence is mostly obtained from cellular and animal models. The process of clinical translation is still constrained by suboptimal target selectivity, dilemmas in bidirectional calcium‑signal regulation, bottlenecks in tissue‑specific delivery and the lack of long‑term safety evaluation. This review is expected to provide theoretical foundations and cutting‑edge insights for novel cardiovascular drug discovery and translational research targeting MAMs‑related calcium homeostasis.

Introduction

Calcium ions (Ca²⁺) serve as important second messengers in cells and exert multiple functions in life activities such as mitochondrial signaling and energy metabolism. Ca²⁺ dynamics represent one of the core mechanisms underlying excitation-contraction coupling (ECC) in cardiomyocytes. A transient elevation in cytosolic Ca²⁺ levels activates the formation of cross bridges, thereby participating in myocardial contraction.1 During contraction, mitochondria accumulate Ca²⁺ and are involved in oxidative phosphorylation as well as the function of the electron transport chain (ETC) for ATP synthesis.2 In these physiological processes, maintenance of Ca²⁺ homeostasis is particularly important: a too-low mitochondrial Ca²⁺ concentration can lead to energy metabolic disturbances, whereas an excessively high concentration may cause calcium overload and trigger apoptosis.3 Relevant studies have revealed that calcium homeostasis imbalance is closely associated with the pathological mechanisms of various cardiovascular diseases (CVDs). For instance, mitochondrial calcium overload has been linked to ox-LDL-induced apoptosis of vascular endothelial cells (VECs), which is considered a pathological origin of atherosclerosis (AS).4 Meanwhile, downstream Ca²⁺ signaling pathways may target complex inflammatory signaling cascades to participate in this process.5 In addition, abnormal increases in mitochondrial Ca²⁺ can lead to decreased mitochondrial membrane potential (MMP), mitochondrial swelling, and release of pro-apoptotic factors such as CytC, which in turn promote excessive opening of the mitochondrial permeability transition pore (mPTP), thereby mediating myocardial ischemia-reperfusion injury.6,7 However, excessively low Ca²⁺ levels are also harmful to the cardiovascular system. This condition forces cardiomyocytes, owing to reduced mitochondrial oxidative activity, to rely on glycolysis for energy supply, consequently promoting the development of cardiac hypertrophy and heart failure (HF).8

The endoplasmic reticulum (ER) is a critical organelle that regulates intracellular Ca²⁺ and maintains Ca²⁺ homeostasis through three modalities: Ca²⁺ uptake, Ca²⁺ storage, and Ca²⁺ transport. Mitochondrial Ca²⁺ homeostasis is closely linked to communication between mitochondria and the ER, which primarily facilitates Ca²⁺ transfer, signal transduction, mitochondrial metabolism, and mitochondrial dynamics.9,10 Recent studies have revealed that, during the regulation of mitochondrial Ca²⁺ homeostasis, mitochondria and the ER not only exhibit functional interactions but also maintain physical connections. Proteins located on the ER membrane can bind to mitochondrial membrane proteins, forming protein-protein complexes at the nanoscale that serve as central hubs for Ca²⁺ homeostasis regulation.11 These connection sites are termed mitochondria-associated ER membranes (MAMs). MAMs form dynamic microdomains maintained by specialized tether and spacer proteins,12 providing a high-Ca²⁺ microenvironment that is essential for mitochondrial internalization of Ca²⁺.10 Recent evidence has confirmed that MAMs maintain mitochondrial Ca²⁺ homeostasis through associated protein bridges. Among these, tethering proteins such as the IP3R/GRP75/VDAC1 complex, MFN1–MFN2, and VAPB-PTPIP51 function as Ca²⁺ transport complexes that regulate Ca²⁺ transfer at MAMs.13–15 These findings highlight the significance of MAMs-mediated Ca²⁺ homeostasis in mitochondrial dysfunction and the pathophysiology of CVDs, and also suggest that modulating MAMs-mediated Ca²⁺ homeostasis may offer novel therapeutic strategies for CVDs.

In recent years, several reviews have investigated the regulatory roles of MAMs in CVDs from multiple dimensions. Chen et al16 focused on the intrinsic association between MAMs and cardiovascular inflammation, and elaborated the molecular mechanisms whereby MAMs mediate inflammatory signaling activation, oxidative‑stress imbalance and immune‑response regulation. By contrast, Ding et al17 reviewed the contributions of dysregulated calcium transport, apoptosis, autophagy and lipid metabolism to the progression of cardiovascular diseases as well as their therapeutic potential, on the basis of the structural characteristics and versatile biological functions of MAMs. Compared with the aforementioned studies, the present review centers on MAMs‑governed calcium homeostasis, systematically dissects key molecular nodes including IP3Rs‑GRP75‑VDAC1, VAPB‑PTPIP51, MFN1‑MFN2 and mitochondrial calcium uniporter (MCU), and compares their disease‑specific alterations in myocardial ischemia‑reperfusion injury, atherosclerosis, HF, pulmonary arterial hypertension and diabetic cardiomyopathy. Relevant intervention strategies are further summarized within the analytical framework of “pharmacological target‑candidate agent‑mechanism of action‑disease‑related evidence”. Moreover, critical translational bottlenecks, such as target selectivity, therapeutic window, tissue‑specific delivery, pharmacokinetic profiles and long‑term safety, are highlighted and analyzed. This review is expected to provide more comprehensive theoretical support for novel‑drug discovery and translational research targeting MAMs‑mediated calcium homeostasis.

MAMs Structure and Protein Complexes in Calcium Homeostasis Regulation

In eukaryotic cells, multiple organelles or membrane systems interact through tightly coupled multiple membrane contact sites (MCS) for material transfer and signal exchange.12,18 Among these, the interactions between mitochondria and the ER account for 5–20% of the total organelle contact regions.19 This structure was first observed in morphological studies of hepatocytes in the early 1970s and was found to be widely present; however, it was initially regarded as an incidental ultrastructural feature.20 It was not until 1990 that MAMs were first isolated and biochemically characterized through gradient centrifugation and subfractionation techniques, confirming that MAMs represent specific membrane contact sites between the ER and mitochondria.21 Of note, mitochondria and the ER are highly dynamic organelles, and the distance at their contact sites—MAMs is not constant, typically ranging from 10 to 30 nm, although recent studies have revealed that the ER–mitochondria distance can extend up to 80 nm.22 Furthermore, the morphological architecture of MAMs is also diverse. In most cells, ER tubules wrap around mitochondria, covering 2% to 5% of the mitochondrial surface area, although partial encirclement of mitochondria by the ER (approximately occupying 50% of the mitochondrial circumference) or complete encirclement has also been observed.12

Proteomic analyses and biochemical studies have revealed that MAMs are not merely simple regions formed by the close apposition of mitochondrial and ER membranes, but rather physical connections established by protein chains that stabilize their architecture and mediate inter-organellar communication.23 Examples of such protein complexes include the inositol 1,4,5-trisphosphate receptors (IP3Rs)-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel (VDAC) complex, the vesicle-associated membrane protein-associated protein B (VAPB)-protein tyrosine phosphatase interacting protein 51 (PTPIP51) complex, homodimers of mitofusin-2 (MFN2)-MFN2 or heterodimers of MFN2-MFN1, as well as the B cell receptor associated protein 31 (BAP31)-mitochondrial fission 1 protein (FIS1) complex.24,25 Collectively, these complexes exert important regulatory functions in various cellular pathophysiological processes, including Ca²⁺ transfer, lipid metabolism, mitochondrial dynamics, ER stress, oxidative stress, apoptosis, and autophagy. Meanwhile, exploratory studies on MAMs tethering proteins are ongoing. Recently, FKBP8 and PDZD8 have also been identified as being closely associated with tethering and lipid metabolism.26 Together, these protein complexes collectively ensure the structural integrity and functional diversity of MAMs. Details are presented in Table 1.

Table 1.

MAMs Protein Complexes Involved in Ca²⁺ Transport and Their Functions

Protein Complex	Protein	Functions and Mechanisms
IP3Rs-GRP75-VDAC127,28	IP3Rs	Located on the ER membrane, regulating ER Ca2⁺ release
	GRP75	Links IP3R to VDAC1, forming a complex that mediates Ca2⁺ transfer
	VDAC1	Located on the outer mitochondrial membrane, mediating Ca2⁺ internalization
VAPB-PTPIP5129,30	VAPB	MAMs tethering proteins that facilitate IP3Rs/GRP75/VDAC1 complex-mediated Ca2⁺ transfer by maintaining the structural integrity of MAMs
	PTPIP51
MFN1-MFN225,31,32	MFN1	MAMs tethering proteins that regulate calcium homeostasis by mediating the contact distance of MAMs
	MFN2

IP3Rs/GRP75/VDAC1 Protein Complex in Calcium Homeostasis Regulation

The IP3Rs/GRP75/VDAC1 protein complex represents the most well-characterized Ca²⁺ transfer unit between mitochondria and the ER. Through ER IP3Rs, MAMs release Ca²⁺ to form local calcium spike microdomains, which in turn stimulate mitochondrial Ca²⁺ uptake.27 IP3Rs exist in three subtypes: IP3R1 is widely distributed in the brain, oocytes, and eggs; IP3R2 is expressed in epithelial, cardiac, and skeletal muscle tissues; and IP3R3 is highly expressed in various cell types and tissues.33,34 Among these, IP3R3 primarily mediates ER Ca²⁺ release and regulates mitochondrial Ca²⁺ uptake, whereas IP3R1 is mainly involved in cytosolic Ca²⁺ mobilization. VDAC1 is located on the outer mitochondrial membrane and mediates Ca²⁺ internalization. GRP75 serves as an adaptor protein enriched in the MAMs gap, bridging IP3Rs and VDAC1 to form a stable ternary complex that mediates Ca²⁺ transfer between the ER and mitochondria. Ultimately, Ca²⁺ enters the mitochondrial matrix through the mitochondrial MCU localized on the inner mitochondrial membrane, thereby participating in cellular signaling regulation.28

In addition, multiple regulatory proteins can interact with the IP3Rs/GRP75/VDAC1 complex to more precisely modulate Ca²⁺ transfer efficiency. For example, Sigma-1 receptor (Sig1R) can bind to Binding Ig Protein (BIP) to form a Ca²⁺-sensitive complex. The Sig1R-BiP complex effectively responds to changes in ER Ca²⁺ levels during signal transduction and prevents IP3R degradation by binding to it, thereby maintaining calcium flux.35 Cyclophilin D (CypD) can directly interact with the VDAC1/GRP75/IP3R1 complex to regulate ER–mitochondrial Ca²⁺ transfer in cardiomyocytes.36 Furthermore, the E3 ubiquitin ligase Parkin can ubiquitinate IP3Rs to promote their proteasomal degradation, precisely regulating complex stability and calcium flux.37 The synergistic effects of these regulatory mechanisms ensure that mitochondrial calcium signals undergo normal fluctuations within a certain range.

Under physiological conditions, the IP3R-GRP75-VDAC1 complex-mediated periodic calcium oscillations deliver Ca²⁺ to the mitochondrial matrix, stimulating mitochondrial oxidative metabolism and ATP production through activation of multiple calcium-dependent dehydrogenases in the tricarboxylic acid cycle, such as pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.28 However, when MAMs architecture is disrupted or Ca²⁺ transport regulation becomes imbalanced, dysfunction of the IP3R-GRP75-VDAC1 complex can directly lead to mitochondrial calcium overload. Excessive Ca²⁺ entry into the mitochondrial matrix can induce opening of the mPTP, leading to the release of reactive oxygen species (ROS) and cytochrome c (Cyt C), ultimately triggering apoptosis or necrosis.38,39

VAPB-PTPIP51 Protein Complex in Calcium Homeostasis Regulation

The VAPB-PTPIP51 complex is an important tethering protein complex between the ER and mitochondria at MAMs. VAPB is an integral membrane protein localized on the ER membrane, while PTPIP51 is a protein located on the outer mitochondrial membrane. Through direct protein-protein interactions, they form a stable complex that bridges the intermembrane gap between the ER and mitochondria, jointly maintaining the physical coupling between the two organelles.40 In terms of Ca²⁺ transport regulation, VAPB-PTPIP51 primarily facilitates Ca²⁺ transfer mediated by the IP3Rs/GRP75/VDAC1 complex by maintaining the structural integrity of MAMs, thereby providing structural support for ER-mitochondrial Ca²⁺ transfer. Studies have shown that overexpression of VAPB or PTPIP51 can enhance ER–mitochondrial signaling, whereas disruption of the VAPB-PTPIP51 connection impairs IP3Rs-mediated Ca²⁺ transfer between the ER and mitochondria.29,30

The Ca²⁺ transport function of the VAPB-PTPIP51 complex depends on the integrity of its structure. It was previously believed that the FFAT domain in the PTPIP51 molecule was critical for its binding to VAPB; however, a recent study has proposed a different view. Using full-length protein-based co-immunoprecipitation, this study demonstrated that FFAT deletion had only a minor effect on VAPB-PTPIP51 binding, whereas mutation or deletion of the coiled-coil domain significantly weakened their interaction and completely abolished the ability of PTPIP51 to promote ER–mitochondria contacts and IP3Rs-mediated Ca²⁺ transfer.41 These findings indicate that the coiled-coil domain of PTPIP51, rather than FFAT, is the critical molecular determinant for its function in ER-mitochondria contacts and calcium signaling. In addition, the connection of the VAPB-PTPIP51 complex is dynamically regulated by multiple upstream signaling pathways. The small GTPase RHOA can directly bind to the ER protein VAPB and regulate the formation of the VAPB-PTPIP51 complex. Genome-wide CRISPRi screening has also confirmed that RHOA is a critical regulator of cellular MAMs levels.40 Other studies have found that glycogen synthase kinase 3β (GSK3β) can reduce ER-mitochondria connections by disrupting the VAPB-PTPIP51 association, and the activity of GSK3β can be inhibited by phosphorylation via the mTORC2/Akt signaling pathway.42

MFN1–MFN2 Protein Complex in Calcium Homeostasis Regulation

The MFN1-MFN2 complex serves as an important structural scaffold that regulates the physical connection between the ER and mitochondria at MAMs, and plays a critical role in both MAMs structural maintenance and calcium signaling regulation. Both MFN1 and MFN2 belong to the Dynamin superfamily of mitochondrial fusion proteins and, together with optic atrophy 1 (OPA1), constitute the core components of the mitochondrial fusion machinery, mediating the fusion of the outer and inner mitochondrial membranes, respectively.43 MFN1 is located on the outer mitochondrial membrane, whereas MFN2 possesses a unique dual-localization property—it is not only localized on the outer mitochondrial membrane but also partially resides on the ER membrane. Through homodimers (MFN2-MFN2) or heterodimers (MFN1-MFN2), these proteins form physical connections that bridge the intermembrane gap at the MAMs interface, anchoring the ER and mitochondria within a nanometer-range contact distance.25,31

The regulation of calcium homeostasis by the MFN1-MFN2 complex is primarily achieved through modulation of the contact distance between the ER and mitochondria at MAMs, as this distance directly affects the efficiency of mitochondrial Ca²⁺ uptake following ER release. Related studies have shown that in MFN2-deficient cells, the distance between the ER and mitochondria is increased, accompanied by a decreased mitochondrial Ca²⁺ uptake capacity.32 Similarly, Chen et al found that MFN2 knockout reduced sarcoplasmic reticulum (SR)-mitochondria contact length by approximately 30% in cardiomyocytes and significantly diminished ER-to-mitochondria Ca²⁺ transfer.44 Conversely, overexpression of MFN2 promotes MAMs formation and enhances ER-mitochondria connections to facilitate Ca²⁺ transfer.45 In addition, MFN2 can directly interact with IP3R3 to mediate ER-mitochondria Ca²⁺ transfer.46 Collectively, these findings indicate that MFN2, by maintaining the physical coupling between the ER and mitochondria, creates the necessary spatial conditions for efficient transfer of IP3Rs-released Ca²⁺ to mitochondria.

The function of the MFN1-MFN2 complex is regulated by multiple post-translational modifications and regulatory proteins. The mitochondrial E3 ubiquitin ligase MITOL can promote ER–mitochondria connections and Ca²⁺ flux by ubiquitinating MFN2.47 The deSUMOylase SENP1 promotes the binding of FIS1 to MFN2 and VDAC1 by regulating FIS1 deSUMOylation, thereby maintaining ER–mitochondria Ca²⁺ transfer.48 MFN2 can also interact with PERK on the ER to inhibit its activity, thereby modulating calcium shuttling and mitochondrial morphology.49 Recent studies have further identified an ER-specific splicing variant of MFN2, termed ERMIT2, which is localized at the ER–mitochondria interface and interacts with mitochondrial MFN2 to anchor the ER and mitochondria, a process that facilitates efficient mitochondrial Ca²⁺ uptake.50 The synergistic effects of these regulatory mechanisms ensure that the MFN1-MFN2 complex can dynamically adjust MAMs structural states and Ca²⁺ transfer efficiency in response to cellular demands.

In the regulation of MAMs calcium homeostasis, the IP3R-GRP75-VDAC1 complex functions as a molecular conduit responsible for the actual delivery of Ca²⁺, the VAPB-PTPIP51 complex serves as a bridging protein that maintains MAMs structural integrity, and the MFN1-MFN2 complex creates the necessary spatial conditions for efficient Ca²⁺ transfer by mediating the physical distance between the ER and mitochondria. Through their division of labor and coordinated actions, these three complexes collectively constitute an efficient MAMs calcium signaling regulatory network.

MAMs-Mediated Calcium Homeostasis and Its Association with CVDs

As a high-energy-consuming organ, the heart relies heavily on precise calcium signaling regulation for its contraction and relaxation. In cardiomyocytes, the SR and mitochondria form close contact microdomains through MAMs, allowing efficient Ca²⁺ transfer that couples excitation-contraction with energy metabolism. However, when the structural integrity or function of MAMs is disrupted, mitochondrial calcium homeostasis imbalance becomes a common pathophysiological substrate underlying various CVDs. Numerous studies have demonstrated that MAMs-mediated calcium homeostasis imbalance is closely associated with the pathological progression of multiple CVDs, including myocardial ischemia/reperfusion (I/R) injury, AS, HF, pulmonary arterial hypertension (PH), diabetic cardiomyopathy (DCM), and cardiac hypertrophy.16,17 The pathological mechanism of MAMs-mediated calcium homeostasis imbalance in cardiovascular diseases is shown in Figure 1.

Figure 1.

Pathological relevance of MAMs-mediated calcium homeostasis imbalance to CVDs.

Notes: (a) Calcium homeostasis; (b)Calcium homeostasis imbalance. In the MAMs calcium homeostasis regulatory network, the IP3R-GRP75-VDAC1 complex functions as a molecular conduit for Ca²⁺ transfer, the VAPB-PTPIP51 complex maintains the structural integrity of MAMs, and the MFN1-MFN2 complex modulates the MAMs contact distance. Disruption of this regulatory network leading to calcium homeostasis imbalance serves as a common pathophysiological basis for various CVDs.

Myocardial Ischemia/Reperfusion Injury

MAMs-mediated calcium homeostasis imbalance exerts a bidirectional regulatory role in I/R injury, and both its pathological effects and protective potential are closely associated with mitochondrial Ca²⁺ transfer efficiency. Under ischemic stimulation, excessive activation of ER IP3Rs/RyRs induces substantial Ca²⁺ release, while SERCA pump activity is inhibited, impairing Ca²⁺ reuptake into the ER, leading to significant cytosolic Ca²⁺ accumulation.51–53 Subsequently, the accumulated Ca²⁺ is efficiently transferred to mitochondria via the IP3R-Grp75-VDAC complex at MAMs, resulting in mitochondrial calcium overload.28 The pathological effects of excessive intramitochondrial Ca²⁺ are mainly manifested in two aspects. On the one hand, high Ca²⁺ concentrations can lead to decreased MMP, mitochondrial swelling, and release of pro-apoptotic factors such as Cyt C. Concurrently, CypD is activated, which not only promotes mPTP opening but also reciprocally enhances Ca²⁺ transfer efficiency of the MAMs complex, forming a positive feedback loop,7,54 ultimately initiating the caspase cascade and inducing apoptosis. On the other hand, the conductance of ETC complexes in mitochondria is enhanced following Ca²⁺-dependent phosphorylation, transiently increasing ATP synthesis. However, the restored energy supply paradoxically triggers excessive contraction, causing necrosis of adjacent cells due to mechanical stress.55

Of note, MAMs-dependent Ca²⁺ transfer may also exert protective effects under certain circumstances. Gӧbel et al reported that perivascular MAMs enrichment promotes remodeling following acute brain injury, whereas MFN2 deficiency attenuates this effect,56 suggesting that moderate mitochondrial Ca²⁺ uptake may help maintain ATP supply under hypoxic conditions, thereby facilitating cardiovascular remodeling and conferring cardiac benefits.

Atherosclerosis

AS is a progressive vascular disease characterized by lipid deposition in the vessel wall and chronic inflammation. Its pathological initiation originates from ox-LDL-induced apoptosis of VECs. Excessive accumulation of ox-LDL can lead to elevated mitochondrial Ca²⁺ and ROS levels, loss of MMP, and release of Cyt C, thereby inducing endothelial cell apoptosis, a process associated with mitochondrial Ca²⁺ overload.4 In addition to inducing VECs apoptosis, MAMs-mediated calcium homeostasis imbalance also affects the functional integrity of VECs. Disruption of Ca²⁺ levels in endothelial cells can compromise endothelial barrier integrity and, by targeting inflammatory signaling pathways such as TLR, NLRP3, and NF-κB, promote the release of inflammatory cytokines and monocyte adhesion, thereby accelerating the formation and progression of atherosclerotic plaques.5,57

In vascular smooth muscle cells (VSMCs), MAMs-mediated ER–mitochondria Ca²⁺ transfer is equally critical. A recent study has revealed a novel MAM-MICU1-MCU signaling axis:58 high-fat and high-cholesterol diets induce increased ER–mitochondria contacts and MAMs formation in VSMCs, promoting ER Ca²⁺ release into mitochondria. Elevated mitochondrial Ca²⁺ binds to mitochondrial calcium uptake 1 (MICU1), thereby weakening its ability to block MCU and leading to sustained MCU opening, ultimately resulting in mitochondrial Ca²⁺ overload. This in turn disrupts fatty acid β-oxidation through regulation of acyl-CoA dehydrogenase (ACADM), leading to lipid deposition. These findings highlight the important role of the MAM-MICU1-MCU axis in AS and the potential therapeutic value of targeting MCU.

Cardiac Hypertrophy and HF

HF represents the end-stage of various CVDs, often resulting from the continuous progression of cardiac hypertrophy, which is a compensatory response of the heart to pathological stimuli such as pressure overload and neuroendocrine activation. MAMs-mediated calcium homeostasis plays a critical regulatory role in this pathological process. Relevant studies have shown that in hypertrophic and failing hearts, ER-mitochondria contact sites exhibit a significant reduction in number and an increase in contact distance, which impairs efficient mitochondrial uptake of Ca²⁺ released from the SR.8,59 Reduced mitochondrial Ca²⁺ levels can further suppress the activity of calcium-dependent dehydrogenases in the tricarboxylic acid cycle, decreasing ATP synthesis and ultimately leading to an energy crisis in cardiomyocytes.60

MAMs-mediated mitochondrial calcium dysregulation in HF involves multiple levels of molecular abnormalities. Among these, the reduction of FUNDC1 is a critical event linking MAMs structural disruption to calcium homeostasis imbalance. FUNDC1 is enriched at MAMs and directly binds to IP3R2 to regulate ER Ca²⁺ release. In HF patients, FUNDC1 expression is significantly decreased, and further studies have revealed that the pathological alterations in cardiac dysfunction are closely associated with suppression of the FUNDC1/MAMs/CREB/Fis1 signaling axis.61,62 Similarly, PACS2, as a MAMs tethering protein, has been demonstrated that its deficiency can disrupt MAMs formation and calcium flux, inhibit mitophagy and energy metabolism, thereby exacerbating hypobaric hypoxia-induced right heart dysfunction.63 Alterations in the composition of the MCU complex also represent an important mechanism. In AngII-induced hypertrophic cardiomyocytes and HF, MCU is overexpressed, whereas silencing MCU can prevent cardiomyocyte hypertrophy by blocking mitochondrial Ca²⁺ overload and ROS generation.64 The recent discovery of the MAMs-resident protein FMO2 provides a new perspective. As a novel component of the IP3R2-GRP75-VDAC1 complex, FMO2 maintains ER–mitochondria contacts and regulates mitochondrial Ca²⁺ signaling through direct binding to IP3R2, thereby ameliorating the progression of HF.65 These multi-level molecular abnormalities provide abundant molecular targets for MAMs-targeted therapy in HF.

Pulmonary Arterial Hypertension

Pulmonary arterial PH is characterized by a progressive increase in pulmonary vascular resistance, and its pathogenesis involves multiple aspects including pulmonary vasoconstriction, vascular remodeling, and right heart dysfunction. In pulmonary arterial smooth muscle cells, MAMs calcium signaling microdomains are critical for maintaining cellular calcium homeostasis and vascular tone. Studies have shown that MFN2 regulates calcium homeostasis by maintaining ER-mitochondria coupling, and downregulation of MFN2 expression can disrupt MAMs and accelerate the proliferation of pulmonary arterial smooth muscle cells, participating in the vascular remodeling process of PH.46 Further studies have demonstrated that MFN2 directly interacts with IP3R3 to mediate mitochondrial Ca²⁺ transfer, and its overexpression can reduce IP3R3 expression, decrease mitochondrial Ca²⁺ transfer, and restore mitochondrial integrity.46,66 In addition, the IP3R2/GRP75/VDAC1 axis can regulate apoptosis by participating in hypoxia-induced mitochondrial Ca²⁺ transfer in pulmonary arterial smooth muscle cells.67 The MCU complex exhibits a unique pattern of dysregulation in PH, where downregulation of MCU expression and upregulation of the inhibitory subunit MICU1 together lead to impaired MCU function, resulting in cytosolic Ca²⁺ overload and mitochondrial Ca²⁺ deprivation.68

MAMs dysfunction can also lead to abnormal mitochondrial Ca²⁺ uptake, increased ROS generation, and ER stress, and these pathological alterations collectively promote abnormal proliferation of pulmonary arterial smooth muscle cells and pulmonary vascular remodeling.66,69 Furthermore, MAMs affect the function of pulmonary VECs through regulation of calcium signaling, and calcium homeostasis dysregulation can disrupt endothelial barrier function and promote inflammatory responses, thereby further aggravating pulmonary vascular injury.16 Recent studies have revealed that the expression of the MAMs structural protein FUNDC1 is significantly reduced in the small pulmonary arteries of PH models, and endothelial cell-specific FUNDC1 deficiency promotes PH development, whereas its overexpression exerts a protective effect.70 Collectively, these findings suggest that restoring MAMs-mediated calcium homeostasis may hold promise for delaying or reversing pulmonary vascular remodeling, offering multi-target intervention strategies for the treatment of PH.

Diabetic Cardiomyopathy

DCM is defined as myocardial dysfunction occurring in diabetic patients independent of valvular heart disease, PH, or coronary artery disease.71 Under hyperglycemic pathological conditions, the structure and function of MAMs undergo abnormal alterations, leading to mitochondrial calcium homeostasis imbalance, which, along with subsequent mitochondrial dysfunction, is considered the underlying cause of cardiomyocyte dysfunction in DCM.72,73 Relevant studies have shown that high-glucose and high-free fatty acid environments can enhance MAMs formation and promote mitochondrial calcium overload. This primarily occurs through inhibition of AMPKα2 activity, which upregulates FUNDC1 expression. FUNDC1 then enhances MAMs formation through interaction with IP3R2, increases mitochondrial Ca²⁺ uptake, and consequently leads to mitochondrial fragmentation and apoptosis.74 The PACS2/IP3R2/FUNDC1/VDAC1 pathway has also been found to participate in high glucose-induced MAMs formation, and activation of this pathway can reduce mitochondrial biogenesis and oxidative phosphorylation.75 In addition, acid sphingomyelinase (ASMase) has been found to be upregulated in diabetic hearts, which can enhance MAMs formation and promote mitochondrial Ca²⁺ overload through MICU1 activation, consequently leading to increased ROS generation, impaired autophagy, and cardiomyocyte apoptosis.73 Therefore, targeting MAMs-mediated calcium signaling pathways, particularly the FUNDC1-MAMs axis and the ASMase-MICU1 pathway, may represent a novel direction for the treatment of DCM.

Pharmacological and Molecular Agents Targeting MAMs-Mediated Calcium Homeostasis

As a core signaling hub for calcium transfer between the ER and mitochondria, MAMs govern mitochondrial calcium homeostasis through their structural and functional integrity. As outlined above, MAMs‑mediated disruption of calcium homeostasis contributes to the pathogenesis of a wide range of cardiovascular diseases. Accordingly, targeting key nodes within the MAMs‑dependent calcium‑signaling cascade has emerged as a cutting‑edge strategy for cardiovascular drug discovery. These nodes encompass IP3R‑driven calcium release from the ER, GRP75‑facilitated tethering, calcium translocation across the mitochondrial outer membrane via VDAC1, MCU‑mediated calcium uptake into the mitochondrial matrix, and feedback crosstalk between endoplasmic reticulum stress and MAMs architecture. Table 2 summarizes drugs and molecular agents targeting MAMs to modulate calcium homeostasis, their underlying mechanisms of action, and a critical appraisal of the supporting evidence.

Table 2.

Evidence‑based Evaluation of Drugs/Molecular Agents Intervening in MAMs to Regulate Calcium Homeostasis

Intervention Target	Compound	Mechanism of Action	Levels of Evidence and Major Models	Target Selectivity and Off‑Target Risk	Safety, Pharmacokinetics(PK)/Delivery and Research‑Development Stage
Targeting the IP3Rs-GRP75-VDAC1 complex	2-APB76,77	Blocks IP3Rs Ca2⁺ channel activity on the ER membrane, inhibiting Ca2⁺ release from the ER to MAMs	In‑vitro: Cardiomyocytes/cardiac tissue; In‑vivo: Mouse myocardial I/R model, rat hyperhomocysteinemia combined with I/R model; Clinical: Not applicable.	Low. In addition to IP3R, it also affects SOCE/Orai and TRP channels, with a concentration‑dependent biphasic effect.	Systemic PK, long‑term toxicity and therapeutic window remain unclear; research‑tool compound/early pre‑clinical stage.
	SB21676378,79	Inhibits GSK3β activity, thereby blocking GSK3β-mediated phosphorylation of IP3Rs, which reduces the stability of the IP3R-GRP75-VDAC1 complex and the frequency of Ca2⁺ channel opening	In‑vitro/ex‑vivo: Hypoxia‑reoxygenation and reperfusion‑related models of cardiomyocytes; Clinical: Not applicable	Moderate‑low. Inhibits GSK3α/β and may broadly affect metabolic, proliferative, and survival pathways.	Cardiovascular PK and long‑term safety are insufficient; mechanism‑based tool/early pre‑clinical stage.
	Targeting peptide (Peptide 4)80	Selectively attenuates ER-to-mitochondria Ca2⁺ transfer by binding to GRP75 and disrupting the IP3R-GRP75 interaction	In‑vitro: Human umbilical vein endothelial cells, macrophages; In‑vivo: ApoE−/− mice fed with western‑type diet; Clinical: Not applicable.	Relatively high. It targets the IP3R‑GRP75 interface, and potential effects on the chaperone function and other interactions of GRP75 need to be ruled out.	Stability, tissue distribution, immunogenicity and repeated‑injection‑related issues need to be addressed; early pre‑clinical lead compound.
Targeting MCU and calcium transport pathways	Ruthenium Red81,82	Blocks Ca2⁺ transmitted via MAMs from entering the mitochondrial matrix by inhibiting MCU, reducing mitochondrial Ca2⁺ accumulation, thereby preventing calcium overload	Ex‑vivo: Isolated rat hearts; In‑vivo: Rat myocardial I/R, pressure‑overload‑induced heart failure; Clinical: Not applicable	Low. It can block multiple types of ion channels and is not a specific inhibitor of MCU.	Poor transmembrane/tissue delivery, non‑linear dose‑response relationship and narrow therapeutic window; research‑tool
	Calcium chelators (BAPTA)38,83	Reduces cytosolic Ca2⁺ concentration by directly binding free Ca2⁺, thereby indirectly inhibiting MAMs-mediated calcium signaling	In‑vitro/ex‑vivo: Myocardial I/R‑related models; In‑vivo: Mice with MI/R treated with BAPTA‑AM nanoparticle platform; Clinical: Not applicable	Very low. It chelates Ca2⁺ globally and affects excitation‑contraction coupling, vascular tone and immune signaling.	Hard‑to‑control BAPTA‑AM activation/distribution; EGTA mainly extracellular; nanoformulation carrier toxicity, scale‑up problems; proof‑of‑concept.
Targeting the ER stress-MAMs-mitochondrial calcium overload axis	4-PBA84,85	Indirectly downregulates IP3R expression and MAMs Ca2⁺ leakage by alleviating ER stress and the UPR	In‑vitro: High‑glycogen atrial myocytes, HL‑1 cells; Evidence of MAMs in cardiovascular animal/clinical studies: Not applicable; Clinical applications have been established for other indications.	Low‑moderate. It indirectly alleviates ER stress and exhibits pleiotropic effects including HDAC inhibition.	Human PK/safety foundation is relatively solid, yet the high dosage and the sodium load of the commonly used sodium‑salt formulation may limit its application in heart failure; pre‑clinical repositioning candidate.
	FL386	Promotes mitochondrial fusion in an MFN1-dependent manner, thereby enhancing ER–mitochondria interactions to optimize Ca2⁺ transfer between the two organelles	In‑vitro: Primary cardiomyocytes; In‑vivo: Mouse MI/R and doxorubicin‑induced cardiotoxicity models; Clinical: Not applicable.	Moderate‑low. It is MFN1‑dependent, while pathways such as prohibitin/STAT3 may constitute alternative mechanisms.	PK, bioavailability and long‑term toxicology data are limited; early pre‑clinical stage.
Targeting MAMs-related signaling pathways	β-Carotene87	Targets STIM1 and IP3R channels, downregulating STIM1 and ORAI1 protein expression, thereby inhibiting the assembly and activation of the IP3R/GRP75/VDAC1-MCU complex	In‑vitro/In‑vivo: Mainly mammary epithelial cells and mouse mastitis models; Evidence of MAMs in cardiovascular research: Not applicable; Human supplement‑related data are available.	Low. It affects multiple pathways including STIM1‑ORAI1 and oxidative stress.	Exposure is affected by diet/formulation; high‑dose administration carries safety warnings for smokers; it is not suitable as a cardiovascular MAMs candidate drug.
	Tanshinone IIA88	Enhances mitochondrial stability, restores MMP, and regulates calcium overload via the METTL3- and SIRT5-mediated UPR pathway	In‑vitro/Genetic‑model‑derived cells: Coronary microvascular endothelial cells; Clinical: Relevant preparation experience exists in some regions, yet no trials targeting the MAMs mechanism have been conducted.	Low‑moderate. METTL3/SIRT5, the UPR and multiple signaling pathways co‑exist.	Poor water solubility, low oral bioavailability and prominent first‑pass metabolism; formulation/delivery‑dependent; MAMs‑related indication remains at the pre‑clinical stage.
	Resveratrol89,90	Regulates MCU in a Zn2⁺-dependent manner, inhibiting mitochondrial Ca2⁺ overload and excessive mPTP opening; inhibits STIM1 phosphorylation to block STIM1-ORAI1 binding, thereby suppressing SOCE	In‑vitro/In‑vivo: Myocardial I/R and multiple cellular Ca2⁺ models; Clinical: Human‑based studies are available, yet MAMs mechanism has not been validated.	Low. It involves multiple pathways, including MCU, STIM1‑ORAI1, IP3R, autophagy and antioxidation.	Low oral bioavailability of the parent drug, rapid metabolism and substantial formulation‑related variability; mechanistic candidate/clinical‑research stage, not approved for cardiovascular MAMs‑related indications.

Targeting the IP3Rs-GRP75-VDAC1 Complex

The IP3Rs-GRP75-VDAC1 complex serves as the core molecular channel mediating ER-to-mitochondria Ca²⁺ transfer at MAMs, and therefore represents the primary target for pharmacological intervention.

2-Aminoethoxydiphenyl Borate (2-APB)

2-APB is a multi-target IP3Rs inhibitor that suppresses Ca²⁺ release from the ER lumen to MAMs by blocking the activity of IP3Rs Ca²⁺ channels on the ER membrane. Relevant studies have shown that in myocardial I/R injury models, 2-APB effectively reversed intracellular and mitochondrial calcium overload and maintained mitochondrial functional homeostasis through intervention in the ITPR1 (IP3R1)/MCU pathway, thereby alleviating myocardial injury.76 Another study also confirmed that in a rat model of hyperhomocysteinemia combined with cardiac ischemia-reperfusion, 2-APB inhibited IP3R-mediated ER–mitochondria Ca²⁺ transfer, significantly reduced infarct size, and improved cardiac function.91

GSK3β Inhibitor SB216763

SB216763 is a selective, ATP-competitive GSK3β inhibitor. By inhibiting GSK3β activity, it blocks GSK3β-mediated phosphorylation of IP3Rs, thereby reducing the stability of the IP3R-GRP75-VDAC1 complex and the frequency of Ca²⁺ channel opening.78,79 Studies have found that in myocardial ischemia-reperfusion injury models, SB216763 treatment decreased the stability of the IP3R-GRP75-VDAC1 complex in cardiomyocytes and reduced ER-to-mitochondria Ca²⁺ transfer, thereby alleviating mitochondrial calcium overload, ROS generation, and apoptosis, exerting cardioprotective effects.79

MAMs-Targeting Peptide (Peptide 4)

Peptide 4 represents a novel strategy targeting MAMs protein-protein interactions. Based on structural and interfacial analysis of the IP3R-GRP75 complex, Ha et al80 designed a cell-penetrating MAMs-targeting peptide, designated Peptide 4. By binding to GRP75 and disrupting the IP3R-GRP75 interaction, this peptide selectively attenuates ER-to-mitochondria Ca²⁺ transfer. This moderate calcium regulation can activate the AMPK-TFEB axis and restore functional autophagic flux. These regulatory mechanisms significantly improved serum lipid profiles, reduced aortic plaque formation, decreased cardiac lipid deposition, and restored MAMs structural normalization in ApoE⁻/⁻ mice, offering a promising intervention strategy for alleviating AS.

Targeting MCU and Calcium Transport Pathways

After Ca²⁺ released from the ER passes through the IP3Rs-GRP75-VDAC1 complex into the intermembrane space of the outer mitochondrial membrane, it must enter the matrix through MCU, which is localized on the inner mitochondrial membrane. Therefore, MCU represents a key target for blocking the terminal pathway of MAMs calcium signaling.

Ruthenium Red

Ruthenium Red is a classic non-specific inhibitor of MCU. By inhibiting the MCU on the mitochondrial matrix side, it blocks Ca²⁺ transmitted via MAMs from entering the mitochondrial matrix and reduces mitochondrial Ca²⁺ accumulation, thereby preventing calcium overload-induced mitochondrial ROS production and oxidative stress. Numerous studies have found that Ruthenium Red treatment significantly reduced reperfusion-induced arrhythmias, atrioventricular block, and mortality following ischemia, while also improving the recovery of post-reperfusion cardiac contractile function and reducing free radical generation. These effects are all dependent on blocking the MCU channel downstream of VDAC1 and cutting off the terminal pathway of calcium flux.81,82 In addition, in pressure overload-induced HF, long-term administration of Ruthenium Red improved cardiac function, attenuated pathological alterations, and preserved mitochondrial integrity.92

Calcium Chelators BAPTA-AM and EGTA

Calcium chelators reduce cytosolic calcium concentration by directly binding free Ca²⁺, thereby indirectly inhibiting MAMs-mediated calcium signaling. These include the cell-permeable BAPTA-AM and the cell-impermeable EGTA. Among these, BAPTA-AM, as a cell-permeable calcium chelator precursor, is hydrolyzed into its active form BAPTA by non-specific esterases after entering the cytosol. By chelating free Ca²⁺, it reduces local Ca²⁺ concentration, thereby indirectly inhibiting the activity of the PERK and IP3R-VDAC1-MCU signaling pathways and alleviating ER stress and mitochondrial damage in cardiomyocytes.83,93 In addition, other studies have confirmed that in myocardial I/R injury, BAPTA-AM alleviated mitochondrial calcium overload and inhibited ischemia-induced ventricular arrhythmias, while BAPTA-AM-loaded targeted nanoplatforms also significantly reduced infarct size and improved cardiac function.77,94,95 Collectively, these studies have demonstrated the potential value of calcium chelation in restoring ER–mitochondria calcium homeostasis and attenuating cardiovascular injury.

Targeting the ER Stress-MAMs-Mitochondrial Calcium Overload Axis

ER stress is an important driving factor for MAMs calcium homeostasis dysregulation. Ca²⁺ depletion within the ER lumen can induce the unfolded protein response (UPR), while ER stress can further exacerbate MAMs Ca²⁺ leakage through upregulation of IP3R expression, forming a vicious cycle.96,97

4-Phenylbutyric Acid (4-PBA)

4-PBA is a chemical chaperone that indirectly downregulates IP3R expression and MAMs Ca²⁺ leakage by alleviating ER stress and the UPR. In BMECs models, 4-PBA pretreatment significantly inhibited the activation of ER stress markers such as BIP and XBP1, reduced ER lumen Ca²⁺ release, and synergistically improved mitochondrial respiratory chain function.84,87 Recent studies have clearly demonstrated that in high glucose-treated primary atrial cardiomyocytes, 4-PBA effectively inhibited the ER stress-MAMs-mitochondrial calcium overload axis, thereby reducing cardiomyocyte death.85 These findings suggest the potential of 4-PBA as a potential intervention agent for metabolic CVDs such as DCM.

Flavagline 3 (FL3)

FL3 is a small-molecule compound derived from natural products. Zhong et al86 demonstrated that FL3 promotes mitochondrial fusion in an MFN1-dependent manner, thereby enhancing ER–mitochondria interactions to optimize Ca²⁺ transfer between the two organelles. FL3 treatment significantly reduced the peak of mitochondrial calcium in primary cardiomyocytes while increasing calcium retention in the MAM region, thereby protecting mitochondrial function under I/R injury conditions and effectively reducing cardiomyocyte apoptosis and infarct size. Of note, compounds of the flavagline family, to which FL3 belongs, have been confirmed to possess broad-spectrum cardioprotective activity. They can not only attenuate doxorubicin-induced cardiomyocyte apoptosis and cardiac fibrosis,98 but also exert cardioprotective effects by targeting prohibitin via the STAT3 signaling pathway,99 positioning FL3 as an effective cardioprotective agent.

Natural Products Targeting MAMs-Related Signaling Pathways

In addition to directly targeting MAMs structural proteins and Ca²⁺ channels, modulating upstream or bypass signaling pathways of MAMs to restore calcium homeostasis also represents an important intervention strategy.

β-Carotene

β-Carotene directly targets the store-operated calcium entry (SOCE) sensor STIM1 and IP3R channels, downregulating STIM1 and ORAI1 protein expression, thereby inhibiting the assembly and activation of the IP3R/GRP75/VDAC1-MCU complex.87 This mechanism does not directly act on the IP3R-GRP75-VDAC1 complex, but rather reduces extracellular calcium influx by inhibiting upstream STIM1, indirectly repairing ER stress-induced calcium homeostasis imbalance. Nevertheless, the existing evidence is predominantly obtained from selected non‑cardiovascular preclinical models. To date, it remains unvalidated whether β‑carotene exerts equivalent regulatory actions within cardiomyocytes or vascular cells, and there is a lack of clinical evidence supporting its application in cardiovascular diseases. Accordingly, β‑carotene is currently better suited as a mechanistic candidate for deciphering the crosstalk between STIM1‑SOCE and MAMs‑dependent calcium signaling, rather than a well‑validated therapeutic agent for cardiovascular disorders.

Tanshinone IIA

Tanshinone IIA is the active component of the Chinese medicinal herb Salvia miltiorrhiza. Pu et al88 found that it primarily exerts its effects through the METTL3- and SIRT5-mediated UPR pathway. Tanshinone IIA treatment enhanced mitochondrial stability, restored MMP, and regulated calcium overload via the METTL3 and SIRT5-mediated UPR pathway. These findings reveal a novel mechanism by which MAMs calcium homeostasis is regulated through the ER-mitochondria UPR signaling axis.

Resveratrol

Resveratrol is a natural polyphenolic compound found in Polygonum cuspidatum and exhibits multi-target protective effects in CVDs. Studies have shown that resveratrol can regulate MCU in a Zn²⁺-dependent manner, inhibiting mitochondrial Ca²⁺ overload and excessive mPTP opening induced by myocardial I/R injury, thereby maintaining mitochondrial homeostasis and exerting cardioprotective effects.89 In terms of calcium signaling regulation, resveratrol has been found to block the binding of STIM1 to ORAI1 by inhibiting STIM1 phosphorylation, thereby suppressing SOCE.90 Meanwhile, resveratrol-induced autophagy has also been shown to depend on IP3R and cytosolic Ca²⁺ signaling.100 Collectively, these studies reveal a multi-target systemic regulatory effect of resveratrol on ER-mitochondria calcium homeostasis.

Discussions and Perspectives

As the core microdomain for physical and functional coupling between mitochondria and the ER, MAMs precisely regulate Ca²⁺ transfer through protein complexes such as IP3Rs-GRP75-VDAC1, VAPB-PTPIP51, and MFN1-MFN2, playing an irreplaceable role in maintaining mitochondrial calcium homeostasis. Structural or functional abnormalities of MAMs can result in insufficient or excessive mitochondrial Ca²⁺ uptake, which subsequently leads to impaired ATP generation, mPTP opening, reactive oxygen‑species accumulation and the activation of apoptotic signaling cascades. These events ultimately promote the initiation and progression of myocardial ischemia‑reperfusion injury, atherosclerosis, HF, pulmonary arterial hypertension, and diabetic cardiomyopathy. Given that physiological mitochondrial Ca²⁺ uptake is essential for oxidative phosphorylation and excitation‑contraction coupling, both calcium deprivation and calcium overload may exert pathogenic effects. Therefore, therapeutic strategies should aim to restore dynamic calcium homeostasis under pathological conditions, instead of chronically and systemically inhibiting or activating a single pathway. Importantly, nearly all therapeutic evidence summarized herein is derived from in‑vitro cellular experiments, ex‑vivo organ models and rodent studies. To date, no clinical investigations in cardiovascular cohorts have established a causal link between MAMs target engagement, the recovery of compartment‑specific Ca²⁺ fluxes and clinical benefits. Human exposure or clinical experience of some compounds for alternative indications may only offer indirect insights into their safety and pharmacokinetic properties, and cannot be construed as clinical evidence for the efficacy of MAMs‑targeted therapies.

Considering mechanistic robustness, target selectivity and druggability, spatiotemporally selective modulation of the IP3R-GRP75 interaction interface represents the most promising strategy for MAMs-targeted therapy. Reversible interface blockers and targeted peptides can precisely regulate local ER-mitochondrial Ca²⁺ transmission while preserving physiological calcium signaling, superior to nonselective IP3R suppression, global calcium chelation, and persistent MCU inhibition. Despite preliminary proof-of-concept, targeted peptides face unresolved limitations in stability, immunogenicity, tissue distribution and lesion delivery. The MCU-MICU1 system acts as a secondary disease-stratified target, yet its context-dependent activity and potential metabolic impairment induced by systemic inhibition restrict its applicability. MFN2, FUNDC1 and PACS2 exhibit disease-specific potential but lack validated druggability and long-term safety profiles. Current pharmacological tools including 2-APB, ruthenium red, BAPTA-AM and EGTA suffer from poor selectivity and broad physiological interference, limiting their utility as mechanistic probes rather than clinical agents. Although 4-PBA has clinical experience in other diseases, its indirect action and absent cardiovascular MAMs-related validation restrict its translational value. Similarly, the multi-target effects and pharmacokinetic properties of FL3 remain insufficiently characterized. Natural products including β-carotene, tanshinone IIA and resveratrol are constrained by non-cardiovascular preclinical evidence, off-target activities, heterogeneous formulations and undefined in vivo exposure. Collectively, target biological feasibility does not guarantee clinical maturity. Further validation in cardiovascular-specific models is essential to clarify target-dependent efficacy, optimal dosage, tissue exposure, pharmacokinetic behavior and long-term safety for future translational applications.

Successful clinical translation prioritizes the construction of a rigorous evidence chain spanning “exposure-target engagement-compartment-specific Ca²⁺ flux-pharmacodynamic efficacy”. First, target-dependent pharmacological effects require rigorous replication and validation in human induced pluripotent stem cell-derived cardiomyocytes, human cardiovascular tissues, and large animal models, with full consideration of confounding comorbidities including age, sex, diabetes mellitus, and renal insufficiency. Second, the development of reversible, controllable delivery systems with precise selectivity for myocardial or vascular lesions is essential to clarify stage-specific intervention strategies, dose-effect correlations, and optimal therapeutic windows. Third, comprehensive systematic evaluation is necessary to assess drug impacts on myocardial excitation-contraction coupling, cardiac electrical conduction and rhythm stability, vascular tone regulation, metabolic adaptation, immune homeostasis, and long-term organ toxicity. Finally, specific pharmacodynamic biomarkers that accurately reflect MAMs structural integrity, subcellular Ca²⁺ dynamics, and target engagement levels should be established to support patient stratification, rational dose escalation, and early therapeutic efficacy assessment. Only after forming a closed evidentiary loop covering mechanistic validation, pharmacokinetic characterization, and safety evaluation can early-phase clinical trials centered on mechanistic endpoints be initiated, followed by systematic assessments of long-term cardiovascular outcomes.

Funding Statement

This work was supported by National Major Science and Technology Project: Research on Interventional Schemes and Mechanisms of Traditional Chinese Medicine in Preventing and Treating Anthracycline-Related Cardiac Injury (Grant No. SQ2023AAA032386), A Clinical Study of Chinese Materia Medica for Preventing anthracycline-induced cardiotoxicity and Replenishing Qi-Nourishing Yin Therapy for Anthracycline-Related Frequent Ventricular Premature Beats (Grant No. 2023ZD0502604), the National Key Research and Development Program (No.2022YFC3500101): Research on Innovative Pathogenesis and Clinical Diagnosis and Treatment Strategies for Coronary Heart Disease-Related Heart Failure, Dongying Natural Science Foundation Health and Wellness High-Quality Development Joint Fund Project (2025ZRWS003), and Dongying City Natural Science Foundation Project (2023ZR032).

Author Contributions

Maoxia Fan is the first author. All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.