Mitochondria-Targeting Accumulation of 5MEF Probe for Early Diagnosis of Myocardial Infarction

Abstract

Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, necessitating the development of more precise diagnostic strategies beyond conventional biomarker detection and imaging techniques, which often suffer from limited specificity and sensitivity. Here, we report the design and evaluation of a mitochondria-targeting accumulation probe, 5MEF, for MI diagnosis. In vitro assays demonstrated that 5MEF exhibits high specificity and selective accumulation in H9c2 cells compared to that in HUVECs. In the rat MI model, 5MEF showed significant differences between the infarcted area and the noninfarcted area, indicating that 5MEF has diagnostic potential. Biosafety assessments conducted in vitro and in vivo revealed no significant cytotoxicity or adverse effects. In conclusion, our findings demonstrate that 5MEF is a highly promising molecular probe for the early detection of MI, providing a highly specific and safe diagnostic tool with a significant potential for clinical translation.

Introduction

Cardiovascular disease has always been a major threat to human health. − According to the authoritative report of the World Health Organization, the number of heart diseases has soared since 2000 and even increased by about 9 million in 2019, resulting in 17.9 million deaths, accounting for 32% of all deaths worldwide. , Myocardial Infarction (MI), the most severe manifestation of coronary heart disease, stems from the abrupt occlusion of coronary arteries, resulting in diminished myocardial blood flow and subsequent ischemic injury. − It is the main cause of death of all cardiovascular diseases and has become a major public health problem that urgently needs to be solved. Consequently, the pursuit of an early, accurate diagnosis coupled with prompt treatment strategies holds paramount importance in saving lives and enhancing patient prognosis.

Cardiac mitochondria, as the “energy factory” in the cell, produce about 6 kg of ATP per day to support the continuous contraction and relaxation of the heart. More importantly, the mitochondrial membrane potential of cardiomyocytes is much higher than that of normal cells, and its homeostasis is closely related to the normal function of the heart. MI, characterized by acute cardiomyocyte ischemia and hypoxia, triggers cell damage, apoptosis, and necrosis. In this process, mitochondria, pivotal in apoptosis regulation and energy metabolism, undergo profound structural and functional alterations, serving as a crucial biomarker for assessing myocardial injury severity and prognosis. − Mitochondria dynamically adapt to cardiomyocyte metabolic fluctuations, modulating their morphology via fission and fusion in response to stressors like hypoxia, ischemia, reperfusion, oxidative stress, or inflammation. , Studies have shown that mitochondrial fission is further enhanced in ischemic or reperfusion cardiomyocytes, accompanied by a decrease in membrane potential and ATP production, ultimately triggering mitophagy to eliminate damaged mitochondria. , At the same time, excessive ROS production and enhanced opening of mitochondrial permeability transition pore (MPTP) can induce myocardial cell necrosis or apoptosis. , Thus, harnessing mitochondria’s intricate regulation of cardiomyocytes during MI and developing efficient, mitochondria-targeted diagnostic tools are paramount for early MI detection, disease progression monitoring, and therapeutic efficacy evaluation.

In recent years, with the rapid development of molecular biology, nanotechnology, and optical imaging technology, fluorescence imaging has shown great application potential in biomedical research due to its advantages of high sensitivity, high specificity, and noninvasiveness. − Notably, in cardiovascular disease diagnosis, the strategic design of specific fluorescent probes has enabled the successful monitoring of crucial myocardial biomarkers, including reactive oxygen species, calcium ion concentrations, and enzyme activities, which provides a powerful tool for in-depth understanding of the pathophysiological mechanism of myocardial infarction. − However, there are relatively few mitochondria-specific fluorescent probes that can effectively diagnose the myocardial infarction area, which limits their wide application in clinical diagnosis.

In this study, we employed 5MEF, a lipophilic cationic small-molecule fluorescent probe previously reported by our group, which exhibits excellent fluorescence properties, high sensitivity, and superior cardiac uptake and contrast, for the diagnostic imaging of early-stage myocardial infarction. Notably, we discovered that 5MEF can accurately identify and label mitochondria within infarcted myocardial tissue by leveraging the elevated mitochondrial membrane potential and MPTP activation, which enhance its affinity for lipophilic cations. This study not only introduces a novel and effective optical-imaging-based strategy for early myocardial infarction diagnosis but also elucidates the pivotal role of mitochondria in the infarction process, thereby providing a theoretical foundation for the development of myocardial protection strategies.

Results and Discussion

To evaluate the targeting and accumulation of 5MEF probes (Figure A) in cardiomyocyte mitochondria in vivo, we used fluorescence microscopy and incubated 5MEF with mitochondrial marker MitoTracker Red in H9c2 (Figure B). Our results demonstrated a strong overlap between the green fluorescence signal of 5MEF and the red mitochondrial marker MitoTracker in H9c2 cells, with a colocalization coefficient of 0.949, indicating excellent mitochondrial colocalization (Figure S1). In contrast, 5MEF exhibited almost no detectable green fluorescence signal in HUVECs, and the corresponding colocalization coefficient was only 0.342 (Figure S2). The fluorescence quantitative results also strongly demonstrated that 5MEF can target cardiomyocyte mitochondria well, indicating that 5MEF has the potential to be used as a novel and promising probe for the assessment of myocardial mitochondrial dysfunction (Figure C).

1.

Mitochondrial targeting and accumulation with 5MEF in vitro. (A) Structure of the 5MEF. (B) Targeting and accumulation of 5MEF to H9c2 and HUVECs (n = 3, scale bar = 20 μm). (C) Analysis of fluorescence quantitative results. (D, E) In vitro cell viability for 12 and 24 h with different concentrations of 5MEF. ***P < 0.001.

We further evaluated the effect of 5MEF on the cell viability. At a concentration of 100 μM, the viability of H9c2 and HUVEC cells remained above 85% after 12 h of incubation, indicating good tolerance under acute exposure. However, after 24 h of incubation, the viability decreased to approximately 70–75%, suggesting a moderate level of cytotoxicity upon prolonged exposure. These results provide a more comprehensive assessment of the short-term biosafety of the 5MEF (Figure D,E).

As illustrated in Figure A, mitochondria normally maintain a high intracellular Ca²⁺ concentration to regulate muscle excitation, contraction, and relaxation. Upon cellular damage, altered permeability of the low-conductance-permeability transition pore (MPTP) leads to Ca²⁺ overload and MPTP activation. At this stage, the fluorescent probe calcein acetoxymethyl ester (Calcein AM) is introduced. As a nonpolar, membrane-permeable dye, it passively diffuses into the cytoplasm and mitochondria, where intracellular esterases hydrolyze it to the polar fluorescent calcein, which remains intracellularly retained. Cobalt chloride (CoCl₂) is subsequently added to quench cytoplasmic fluorescence. Under physiological conditions, the MPTP remains closed, preventing CoCl₂ from entering the mitochondria and thus preserving mitochondrial fluorescence. In damaged cells, MPTP opening permits CoCl₂ entry, resulting in partial mitochondrial fluorescence quenching in addition to cytoplasmic quenching.

2.

Assessment of the MPTP opening following MI. (A) Schematic illustration of MPTP opening. (B) Opening of MPTP in H9c2 of the nonhypoxia group. (C) Opening of MPTP in H9c2 of hypoxia group (n = 3, scale bar = 25 μm). (D) Exploration of the mechanism of entry of 5MEF into H9c2 after MI. The nuclei were labeled with blue fluorescence; 5MEF were labeled with green fluorescence; Mitochondria were labeled with red fluorescence (n = 3, scale bar = 25 μm). (E) Assessment of mitochondrial membrane potential dynamics.

To investigate the opening status of MPTP postmyocardial infarction, we utilized calcein AM as a fluorescent cell staining agent specifically designed for live cells. Calcein AM readily permeates viable cell membranes and, upon entering the cytoplasm, is hydrolyzed by esterases into calcein, which remains within the cells and emits a robust green fluorescence. Our experimental results (Figure B,C) demonstrated that in normal cardiomyocytes, where MPTP remains closed, the cytoplasm, including mitochondria, exhibited intense green fluorescence. Upon the addition of CoCl₂, it is noteworthy that calcein itself functions as a metal-chelating indicator, and its fluorescence is quenched when it chelates metal ions such as Co²⁺ at physiological pH. Since CoCl₂ is unable to penetrate mitochondria, our findings showed that calcein in the cytosolic compartment was quenched, while calcein within mitochondria continued to emit green fluorescence. In contrast, H9c2 subjected to hypoxic treatment exhibited a complete disappearance of the green fluorescent signal. This observation indicates that hypoxic stress induced cellular damage, altering MPTP permeability, resulting in calcium overload and subsequent activation of MPTP. When calcein AM and CoCl₂ were introduced to these damaged cells, to which MPTP had opened to some extent, CoCl₂ gained access to mitochondria, causing the quenching of calcein fluorescence within them.

To elucidate mitochondrial membrane potential (ΔΨm) alterations in cardiomyocytes following MI and the potential mechanism of 5MEF intracellular entry, we designed a series of experiments. Mitochondrial membrane potential was assessed using the JC-1 fluorescent probe, with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) serving as a positive control. Hypoxic treatment of H9c2 cells was employed to simulate the post-MI microenvironment. As shown in Figure E, unlike the prominent red fluorescence observed in the control group, the CCCP-treated and hypoxia-treated groups exhibited a marked loss of red fluorescence, accompanied by an intense green signal, indicative of mitochondrial membrane potential depolarization. Consistently, Figure D demonstrates that normal H9c2 cells exhibited the strongest red fluorescence, reflecting intact mitochondrial membrane potential, thereby confirming that 5MEF uptake into mitochondria is dependent on the membrane potential integrity.

In contrast, in CCCP-positive controls, the presence of 5MEF was barely detectable in H9c2 due to the successful disruption of mitochondrial membrane potential, which further validates the critical role of mitochondrial membrane potential in the entry of 5MEF into mitochondria.

In the hypoxia-treated experimental group, although the mitochondrial membrane potential was reduced or even disappeared, we could still observe a weakened green 5MEF fluorescence signal compared with the control group. This finding suggests that under hypoxic conditions mimicking MI, 5MEF is somehow able to enter cardiomyocytes despite impaired mitochondrial membrane potential.

To provide insight into the mechanism of entry of 5MEF into H9c2 after MI, we further introduced cyclosporin A (CsA), a specific inhibitor that closes the MPTP opening. The experimental results showed that the amount of 5MEF entering mitochondria was significantly reduced in response to CsA intervention. This finding prompted us to speculate that after MI, cardiomyocytes are damaged by oxidative stress, and the mitochondrial membrane potential is subsequently reduced, but MPTP opening may occur at this time. Therefore, we reasoned that it may be through this open MPTP pathway that 5MEF enters the cardiomyocytes. This finding not only advances our understanding of the pathological mechanism of MI but also provides new potential targets for future therapeutic intervention.

MI was surgically induced in rats via permanent ligation of the left anterior descending coronary artery, while sham groups underwent identical procedures without coronary occlusion. At 24 h postoperation, transthoracic echocardiography was systematically performed to validate model efficacy (Figure A). Quantitative analysis revealed significant impairment of left ventricular systolic function in MI rats compared to the sham groups. LVEF was markedly reduced in the MI group (42.3% ± 5.1% vs 68.9% ± 4.7%, P < 0.001), and LVFS demonstrated comparable deterioration (18.6% ± 3.2% vs 35.4% ± 3.8%, P < 0.001) (Figure B,C). These results confirmed the successful establishment of the MI model.

3.

Measurement of cardiac function in MI rats. (A) Representative M-mode ultrasound images of rats in the sham and MI groups. (B) Quantitative analysis of the LVEF. (C) Quantitative analysis of LVFS. (n = 4) ***P < 0.001.

The probes were injected into the tail vein of rats at different infarction time points, and the main tissues and organs of rats were collected 1 h later for fluorescence imaging analysis of isolated organs. The experimental results confirmed the specific targeting and accumulation of the 5MEF probe to myocardial tissue (Figure A–C). At different time points, the fluorescence intensity of MI hearts was always lower than that of sham groups, which is consistent with the inflammatory response and oxidative stress in MI, which aggravate mitochondrial damage and dysfunction and reduce membrane potential. In addition, while longitudinal monitoring demonstrated a marginal temporal increase in whole-heart fluorescence intensity within the MI group, one-way ANOVA analysis confirmed the absence of statistically significant differences across time points (Figure D). We speculated that the gradual accumulation of 5MEF probes in the infarcted area was due to aggravated late-stage myocardial infarction and blood stasis, likely resulting from long-term infarction–induced circulatory disorders that hindered rapid clearance by hepatic and renal metabolism.

4.

Myocardial Infarction Targeting and Accumulation with 5MEF. (A–C) Quantification of major tissue fluorescence signals at different time points. (D) 5MEF targeted and accumulation fluorescence imaging of major tissues. From top to bottom, they are 6, 24, and 72 h, respectively. (E) Local targeted fluorescence signals were observed in cardiac sections. From top to bottom, they are 6, 24, and 72 h, respectively. The red and white boxes represent distal noninfarcted and at-risk infarcted areas, respectively. (n = 4–6) ***P < 0.001; **P < 0.01; *P < 0.05.

In order to further demonstrate the targeting and accumulation ability of the probe to the mitochondria of cardiomyocytes, we performed sections of cardiac tissues (Figure E). The results showed that the 5MEF probes in the sham group were uniformly distributed in the myocardial tissue, and the fluorescence signal intensity at different time points was higher than that in the MI group, which was consistent with the conclusion of global cardiac fluorescence imaging. In addition, the fluorescence signal intensity in the proximal region of coronary artery ligation was higher than that in the distal apical region and the anterior wall region, which may be related to the disruption of the distal blood supply and infarction. This irreversible damage leads to the opening of local MPTP, a significant decrease in membrane potential, and mitochondrial disintegration. In summary, the 5MEF probe is a novel and effective tool for the study of myocardial mitochondrial dysfunction.

To determine the safety of the 5MEF probe in vivo, rat blood and major organs were procured postprobe administration. As with the PBS group, the 5MEF probe elicited no discernible pathological alterations in the heart, liver, spleen, lung, or kidney, nor did it induce inflammation or hemorrhage (Figure A). Additionally, no statistically significant differences were observed in serum levels of liver enzymes (ALT, AST) or renal biomarkers (Cre, Ure) between the two groups (Figure B–E). Common blood cells, such as white blood cells, lymphocytes, neutrophils, and monocytes, were also within the normal range (Figure F). These findings underscore the favorable biosafety and promising clinical translation potential of the 5MEF probe. We have performed additional histological analyses of major tissues at 72 h postinjection. The results showed no significant tissue damage, further supporting the in vivo biocompatibility of the 5MEF (Figure G–K).

5.

Twenty-four and 72 h safety evaluation in vivo (A–F: 24 h, G–K: 72 h). (A) The main tissues and organs were stained with HE (n = 3, scale bar = 100 μm). (B, C) Quantitative analysis of serum liver markers (n = 3). (D, E) Quantitative analysis of serum renal markers (n = 3). (F) Quantitative analysis of common blood cells (n = 3). (G) The main tissues and organs were stained with HE (n = 3, scale bar = 100 μm). (H, I) Quantitative analysis of serum liver markers (n = 3). (J, K) Quantitative analysis of serum renal markers (n = 3).

Conclusions

This study successfully synthesized a highly efficient fluorescent probe specifically targeting myocardial mitochondria and accumulation, which was rigorously validated for its exceptional specificity and sensitivity in detecting myocardial injury within a myocardial infarction model. This probe presents a novel and promising avenue for early myocardial infarction diagnosis, offering fresh insights into the pathophysiological mechanisms underlying this condition. In addition, the excellent performance of the probe indicates its wide application potential in the diagnosis and treatment of cardiovascular diseases and lays a solid foundation for future clinical translational research.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00098.

• All of the experimental methods include probe preparation, construction of myocardial infarction models, as well as details of in vitro and in vivo cell and animal experiments (PDF)

○.

Q.D., Y.B., and C.D. contributed equally to this work. All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Q.D., Y.B., C.D., S.H., K.Q., and X.W. The first draft of the manuscript was written by Q.D., and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

The authors declare no competing financial interest.