miR-873-5p Suppression Reinvigorates Aging Mesenchymal Stem Cells and Improves Cardiac Repair after Myocardial Infarction

Abstract

Aging poses obstacles to the functionality of human mesenchymal stem cells (MSCs), resulting in a notable decline in their valuable contribution to myocardial infarction (MI). MicroRNAs (miRNAs) play a pivotal role in governing MSC aging; nonetheless, the specific mechanisms remain puzzling. This research delved into the value of miR-873-5p in the management of MSC aging and investigated whether the restraint of miR-873-5p could regenerate aged MSCs (AMSCs), thereby enhancing their healing success for MI. In this study, MSCs were isolated from both young donors (referred to as YMSCs) and aged donors (referred to as AMSCs). The senescence status of these MSCs was evaluated through the application of age-related β-galactosidase (SA-β-gal) staining. Following this assessment, the MSCs, including those treated with anti-miR-873-5p-AMSCs, were then transplanted into the hearts of Sprague–Dawley rats experiencing acute myocardial infarction. Increasing miR-873-5p levels in YMSCs resulted in elevated cellular aging, whereas reducing miR-873-5p expression decreased aging in AMSCs. Mechanistically, miR-873-5p inhibited autophagy in MSCs through the AMPK signaling pathway, leading to cellular aging by suppressing the Cab39 expression. Partial alleviation of these effects was achieved by the administration of the autophagy inhibitor 3-methyladenine. Grafting of anti-miR-873-5p-AMSCs, by enhancing angiogenesis and bolstering cell survival, led to an improvement in cardiac function in the rat model, unlike the transplantation of AMSCs. miR-873-5p which serves as a pivotal element in mediating MSC aging through its regulation of the Cab39/AMPK signaling pathway. It represents an innovative target for revitalizing AMSCs and enhancing their heart-protective abilities.

Introduction

Acute myocardial infarction (AMI) is distinguished by myocardial necrosis emanating from severe and lengthy hypoxia and ischemia in the coronary arteries.¹ Although significant advancements have been made in treatments, such as pharmaceutical therapy and percutaneous coronary intervention, AMI continues to be a leading cause of both mortality and morbidity among the elderly population worldwide. The utilization of mesenchymal stem cells (MSCs) in therapy has garnered considerable attention as a promising and emerging approach for treating AMI in recent years.² Prior research has demonstrated that MSC transplantation possesses the ability to mitigate heart renovation and boost the recuperation of heart function post-AMI. This is achieved by reducing cardiomyocyte apoptosis, promoting cardiomyocyte regeneration, and enhancing angiogenesis.³ Nevertheless, as individuals age, the activity of MSCs significantly declines, characterized by heightened cellular senescence, diminished proliferative capacity, and compromised paracrine secretion functions. Consequently, the cardioprotective potential of MSCs following AMI is significantly hampered.⁴ Hence, there is a pressing need to explore innovative approaches to rejuvenate aging MSCs (AMSCs) with the aim of enhancing their therapeutic potential for elderly patients with AMI.⁵

MicroRNAs (miRNAs) are highly conserved, single-stranded, short RNAs ranging from 20 to 24 nucleotides in length, and they do not code for proteins.⁶ miRNAs exert their regulatory influence on gene expression by binding to the 3′ untranslated regions (UTRs) of targeted mRNAs. They can either facilitate mRNA degradation or inhibit translation. miRNAs play a crucial role in governing a range of life functions within stem cells, such as cell division, survival, and differentiation.⁷⁻⁹

Numerous pieces of literature have substantiated the critical role of miRNAs in governing cell aging in MSCs.¹⁰⁻¹² Hong et al. proposed that inhibiting miR-155-5p could potentially regenerate AMSCs and optimize their treatment outcomes for AMI.¹³ Furthermore, the suppression of miR-34a has the potential to decelerate the aging of cells in MSCs by targeting NAMPT and orchestrating the NAD-Sirt1 pathway.¹⁴ Additionally, our prior research has demonstrated that miR-873-5p mimics can impede AMPK phosphorylation.¹⁵ miR-873-5p is known to be involved in various signaling pathways.^16,17 The deceleration of MSC senescence has been linked to the activation of the AMPK signaling pathway.^18,19 The exploration of a suspected relationship between miR-873-5p and MSC aging is prompted by the observed alteration in miR-873-5p expression levels. Nevertheless, it remains uncertain whether or in what manner miR-873-5p influences the senescence of MSCs in the context of AMI.

Autophagy serves a crucial role in maintaining intracellular homeostasis by eliminating harmful cytoplasmic components through autolysosomes.²⁰ Recent studies have revealed that stem cells, including MSCs, experience cellular senescence at an early stage, a phenomenon closely associated with impaired autophagy function.^21,22 MSCs obtained from older donors exhibited compromised autophagy capabilities. Macrophage migration inhibitory factor, by inducing autophagy, has the potential to refresh these aged MSCs. However, the exact mechanisms by which miR-873-5p may regulate autophagy to induce MSC senescence and the underlying processes involved remain unclear.

This study delved into the underlying molecular pathways, shedding light on the task of miR-873-5p in governing the MSC aging process. Furthermore, it was investigated whether the hindrance of miR-873-5p would be able to potentially resuscitate AMSCs and enhance cardiac protection when these rejuvenated AMSCs were engrafted into a rat model of AMI.

Methods

Ethical Statement

Approval for all animal experiments was granted by the Animal Research Committee of Xuzhou Clinical School, Xuzhou Medical University, in compliance with the guidelines set forth by the NIH for the Care and Use of Laboratory Animals. Throughout the course of this study, we scrupulously upheld ethical principles. Anesthetics were consistently administered during all procedures to minimize any discomfort, and the rats were sedated via an intraperitoneal injection of pentobarbital sodium (50 mg/kg).

Cells Culture

Human bone marrow samples were acquired from both aged and young donors and extracted from the posterior superior iliac spine. The study of human subjects was reviewed and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (approval number: KY-D-2021-401-01). The MSCs utilized in the study were all within passages 4–6. The human MSCs were sourced from the bone marrow and cultivated in a humidified environment (37 °C, 5% CO2), with the addition of epidermal growth factor (EGF; 5 ng/mL; PeproTech, AF-100-15), fetal bovine serum (10%; Life Technologies, 16,000), and fibroblast growth factor 2 (FGF2; 5 ng/mL; PeproTech, 100-18B). For both YMSCs and AMSCs, three separate cell platings of equal cell quantities were carried out at 3 day intervals.

Differentiation Capacity of MSCs

The MSCs were prompted to undergo differentiation into adipocytes and osteocytes. 6-well plates were used to seed YMSCs and AMSCs, and they were cultivated until they reached complete confluence. Subsequently, the MSCs were immediately exposed to adipogenic or osteogenic conditions by replacing the culture medium with adipogenic (HUXXC-90031) or osteogenic (HUXXC-90021) condition medium obtained from Cyagen. All media changes were performed every 3 days. After a 3 week culture period, both osteocytes and adipocytes were fixed using a 4% paraformaldehyde solution. Adipogenic differentiation of MSCs was affirmed by Oil-red O staining, while osteogenic differentiation was verified by the utilization of Alizarin red solution.

Clonogenic Assay of MSCs

Each well of 6-well culture plates received YMSCs and AMSCs at a density of 500 cells. Following a 14 day period for colony formation, the plates were subjected to staining using a 0.5% crystal violet solution, and the count of observable colonies was performed.

Senescence-Associated β-Galactosidase (SA-β-gal) Assay

To gauge cellular aging in MSCs, SA-β-gal staining was carried out with the Beyotime kit (C0602). MSCs subjected to various treatments were cultured in six-well plates. For SA-β-gal staining, MSCs were fixed for 20 min, followed by three PBS washes; afterward, the staining solution was applied, and the samples were incubated at 37 °C overnight. Subsequently, randomly selected MSCs displaying a blue stain, indicative of SA-β-gal positivity, were photographed. By dividing the positive MSC count by the total MSC count in all five visual fields, we determined the percentage of matured MSCs. A minimum of three repetitions were performed for these experiments.

Preparation of MSCs-Derived CdM

The process began with the placement of YMSCs and AMSCs in 6-well plates containing growth media, followed by cultivation until they reached a confluence between 70 and 80%. After various treatments were performed, the growth media in each well were traded out for 2 mL of medium devoid of serum. After a 2 day culturing period, the conditioned medium (CdM) was collected, filtered, and centrifuged, and afterward, they were frozen at −80 °C for future applications.

Assay for HUVEC Tube Formation

To evaluate the angiogenic potential of CdM derived from MSCs, a capillary tube formation assay was conducted. The procedure started with the initial coating of a 96-well plate using growth factor-reduced Matrigel, followed by the introduction of 30,000 HUVECs per well (BD Biosciences, 356230). Following a 6 h treatment period, capillary-like tube formation was visualized through imaging. ImageJ software was utilized for quantifying the number of branches and the dimensions of the tubes. Each experiment was replicated a minimum of three times.

PCR

RNase-free DNase I (Takara, 2270A) was used to treat the total RNA from both MSCs and serum. For the synthesis of reverse transcription primers, the PrimeScript RT Reagent Kit (Takara, RR037A) was employed. To assess mature miR-873-5p’s expression amounts (002623, Applied Biosystems), Taqman miRNA assays were utilized. U6 (002623, Applied Biosystems) was used as a reference for establishing a baseline for relative microRNA expression. The tests were performed on the 7500 Fast Real-Time System (Applied Biosystems), beginning with an initial incubation at 95 °C for 30 s, followed by 40 cycles of 8 s at 95 °C and 30 s at 60 °C. miR-873-5p expression was adjusted to match that of U6 by employing the 2^–ΔΔCt cycle threshold method. A minimum of three independent experiments were conducted.

Western Blot

The cells were lysed in 1 mL of lysis buffer at 4 °C for 30 min, with both buffers containing protease inhibitors from Cell Signaling Technology and Calbiochem, respectively. Protein content in the samples was gauged using the BCA protein assay kit (Pierce, USA). Western blotting followed standard techniques, as previously reported. The following antibodies were employed: phosphorylated-AKT (p-AKT) (44-621G, Thermo Fisher), AKT (4691, Cell Signaling Technology); cleaved caspase-3 (PA5-114687, Thermo Fisher), Bcl-2 (ab196495, Abcam), vascular endothelial growth factor (VEGF, ab52917, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, abcam), calnexin (ab22595, abcam), and horseradish peroxidase-conjugated secondary antibody (Biosharp). Enhanced chemiluminescence reagents were used to visualize the bands, and analysis was performed using the iBrightCL1000 gel documentation system (Invitrogen) along with Image Lab Software version 3.0. The bands were detected and subsequently analyzed using enhanced chemiluminescent reagents and the iBrightCL1000 gel documentation system (Invitrogen) in conjunction with Image Lab Software version 3.0.

miR-873-5p Mimic and Inhibitor Transfection

miR-873-5p mimics, inhibitors, and a negative control (miR-control) were procured from GenePharma. To outline the procedure, 1 × 10⁶ MSCs were initially seeded in a 10 cm plate. Transfection of miR-873-3p mimics (50 nmol/L), miR-873-3p inhibitors (100 nmol/L), and their negative controls (50–100 nmol/L) were carried out using Lipofectamine 2000 (Invitrogen, USA) according to the instructions of the manufacturer. Following transfection, the MSCs were cultured at 37 °C with 5% CO₂ for a period of 48 h before being harvested for analysis. It is noteworthy that a minimum of three separate transfection experiments were conducted as part of this study.

Luciferase Assay

The 3′-UTR of human Cab39 was inserted into the pGL3 luciferase reporter vector from Promega, located in Madison, WI, USA. Through overlap extension PCR, alterations were introduced into the seed region of the Cab39 3′-UTR miR-873-5p-binding site. Afterward, miR-873-5p mimics were introduced into 293T cells through co-transfection in 24-well plates, a scrambled miRNA control, or the wild-type or mutant pGL3-Cab39-3′-UTR, all using Lipofectamine 2000 (Invitrogen, 11668027). After 48 h of transfection, luciferase activity was evaluated using the Dual-Luciferase Reporter Assay System Kit (E1910, Promega) per the guidelines of the manufacturer’s instructions. This experimental setup was replicated in a minimum of three independent experiments.

Establishing an MI Model and Evaluating Cardiac Function

Rats of the male Sprague–Dawley strain, within the age range of 6–8 weeks, were obtained from the animal facility at Nanjing Medical University in Nanjing, China. Prior to experimentation, the rats were subjected to orotracheal intubation for mechanical ventilation, following the administration of 50 mg/kg of sodium pentobarbital. To induce cardiac ischemia, a 1.5 mm ligation was surgically placed at the lower edge of the left auricular lobe to obstruct blood flow to that specific area of the heart. After a period of 28 days following exosome injection, the assessment of heart function was carried out using transthoracic echocardiography and a high-resolution microimaging device known as VEVO 2000.

For assessing cardiac function, M-mode imaging was employed with a 30 MHz transducer, focusing on the short axis of the left ventricle. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were determined using the Vevo2000 workstation program.

Staining with Masson Trichrome and Hematoxylin–Eosin

The heart tissue was carefully harvested, stabilized, and then sliced into precise 5 μm sections. As described previously (ref (23)), Mason’s trichrome staining was conducted. To compute the proportion of the infarcted area relative to the total left ventricular (LV) area, the infarcted area was subtracted from the total LV area, and the result was multiplied by 100%. The quantity of inflammatory cells was assessed using hematoxylin–eosin (HE) staining.

Immunofluorescence

Immunofluorescence was carried out as described previously.²⁴ In brief, heart tissue was extracted, fixed, embedded, and sectioned. These sections were subsequently stained using CD31 antibodies (AF3628-SP, Biotechne) and primary antiactin antibodies (MAB1420, Biotechne). Counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.) was carried out for the nuclei.

Immunohistochemistry

Immunohistochemical staining of heart tissue slices using anti-α-SMA (1:100; ab5694, Abcam) and anticleaved caspase3 (1:100; ab19898, Abcam) allowed us to compare the vascular densities of several groups of hearts. Quantifying the frequency with which fields include blood vessels positive for α-SMA was used to represent the arteriole densities.

TUNEL Staining

As previously described in the documented literature,²⁵ TUNEL labeling was employed to evaluate cardiomyocyte apoptosis within cardiac tissue across different experimental groups. Following the staining procedure, fluorescent microscopy images were captured from the tissue slices, which had been counterstained with DAPI. The quantification of apoptosis was achieved by calculating the ratio of TUNEL-positive cells to DAPI-positive cells and subsequently multiplying it by 100%.

Analytical Statistics

For each data point, the mean ± standard deviation was reported. A two-tailed test was employed to assess the differences in mean values between the two groups, and for the analysis of variance among multiple groups, a single-factor design was utilized. The data analysis was accomplished using SPSS (version 17.0, SPSS Inc., Chicago, IL, USA). A significance level of less than 0.05 (P < 0.05) was deemed statistically meaningful.

Results

Characterization of Young and Aged MSCs

In this study, we derived MSCs from both young and old donors extracted from bone marrow. To assess their differentiation potential, we utilized Oil Red staining and Alizarin Red staining, which allowed us to examine the adipogenic and osteogenic capabilities of YMSCs and AMSCs, respectively. Both YMSCs and AMSCs demonstrated the ability to differentiate into adipocytes and osteocytes. However, interestingly, AMSCs exhibited an enhanced adipogenic capacity, while their osteogenic capacity was diminished compared to YMSCs. This suggests that certain functions of AMSCs were compromised, indicating that aging has an impact on MSCs’ differentiation potential (Figure 1a,b). Furthermore, as changes in differentiation potential are indicative of MSCs’ senescence, we assessed cellular senescence in both young and old MSCs. Initially, we conducted age-related β-galactosidase (SA-β-gal) staining, which revealed higher SA-β-gal activity in AMSCs compared to that in YMSCs (Figure 1c). Additionally, Ki67 immunostaining indicated that the proliferation capacity of AMSCs was inferior to that of YMSCs (Figure 1d). Neovascularization promotion is a key aspect of MSC-based treatment for myocardial infarction (MI). Therefore, the angiogenic capability of the conditioned medium (CdM) sourced from YMSCs and AMSCs was analyzed. Figure 1e illustrates that CdM from YMSCs significantly promoted tube formation compared to that from AMSCs. AMSCs-CdM treatment, in contrast to YMSCs-CdM treatment, displayed a reduced endothelial network formation ability (Figure 1e). Moreover, Western blotting analysis indicated that older MSCs expressed higher levels of senescence-related markers, specifically p21 and p53, when compared to younger MSCs. To summarize, these findings indicate that AMSCs manifest an accelerated cellular aging stage (Figure 1g).

Figure 1.

Characterization of young and aged MSCs. (A) Adipogenic differentiation was assessed through Oil Red staining, and the efficiency of adipogenic quantification was evaluated in both YMSCs and AMSCs. Scale bar = 100 μm. (B) Osteogenic differentiation was determined using Alizarin Red staining, and the efficiency of osteogenic quantification was assessed in YMSCs and AMSCs. Scale bar = 100 μm. (C) SA-β-gal staining was performed, and quantitative analysis of SA-β-gal-positive cells was carried out in YMSCs and AMSCs. Scale bar = 200 μm. (D) Immunostaining for the proliferation marker Ki67 was conducted, and quantitative analysis of Ki67-positive cells was performed in YMSCs and AMSCs. Scale bar = 100 μm. (E) Clonogenic assay images demonstrated the proliferative capacity of both YMSCs and AMSCs. (F) Tube formation and tube length analysis in HUVECs treated with YMSC-CdM or AMSC-CdM were conducted. Scale bar = 100 μm. (G) Western blotting was performed, and quantitative analysis of the expression levels of p53 and p21 was carried out in YMSCs and AMSCs. Data are presented as the mean ± SEM n = 3. **p < 0.01; ****p < 0.0001.

miR-873-5p Regulates MSCs Cellular Senescence

In this segment of the study, we conducted qRT-PCR to assess the levels of miR-873-5p in blood samples gathered from both young and elderly volunteers. Our primary focus was to scrutinize the hypothetical significance of miR-873-5p in regulating the MSC aging phenomenon. First, we noted a substantial rise in miR-873-5p portions in older donors compared to their younger counterparts (Figure 2a). This indicated a notable increase in miR-873-5p expression in the elderly group. Furthermore, our investigation revealed a positive correlation between miR-873-5p levels and the cellular senescence of MSCs. Specifically, miR-873-5p levels were substantially higher in AMSCs compared to YMSCs, highlighting a noticeable connection between miR-873-5p and the MSC aging process (Figure 2b). To delve into the role of miR-873-5p in the aging process of MSCs, we conducted experiments involving the use of miR-873-5p mimics to treat YMSCs. This treatment resulted in a considerable uptick in miR-873-5p levels. Importantly, when juxtaposed with the miR-control procedure, miR-873-5p mimic administration resulted in a notable enhancement in the expression levels of p21 and p53 proteins, along with an increase in SA-β-gal activity in YMSCs (Figure 2c,d). These findings strongly support the idea that miR-873-5p is involved in promoting cellular senescence in YMSCs. Furthermore, to solidify the position of miR-873-5p in regulating MSC aging, we administered miR-873-5p inhibitors to AMSCs. Following this treatment, there was a substantial reduction in miR-873-5p levels, SA-β-gal activity, and the presence of p21 and p53 in AMSCs (Figure 2e–f). This suggests that inhibiting miR-873-5p can mitigate the senescence processes in AMSCs. Overall, our data strongly support the notion that miR-873-5p is intricately entangled in mediating the aging of MSCs.

Figure 2.

miR-873-5p mediated the cellular senescence of MSCs. (A) Expression of miR-873-5p was assessed in serum samples from both aged and young donors. (B) miR-873-5p expression levels were detected in both AMSCs and YMSCs. (C) Images of SA-β-gal staining and quantitative analysis of SA-β-gal-positive YMSCs were conducted following transfection with either miR-control or miR-873-5p mimic. Scale bar = 100 μm. (D) A Western blot assay was performed, and quantitative analysis of p53 and p21 expression was carried out in YMSCs transfected with either miR-control or miR-873-5p mimic. (E) SA-β-gal staining images and quantitative analysis of SA-β-gal-positive AMSCs were obtained following transfection with either miR-control or miR-873-5p inhibitor. Scale bar = 100 μm. (F) Western blotting was conducted, and quantitative analysis of p53 and p21 expression was performed in AMSCs transfected with either miR-control or miR-873-5p inhibitor. Data are presented as the mean ± SEM n = 3. **p < 0.01, ***p < 0.001.

miR-873-5p Suppression Reinvigorates Aged MSCs Via Inducing Autophagy

Recent investigations have shed light on the suppressing function of autophagy in cellular senescence. In the context of aged mesenchymal stem cells (MSCs), we observed evidence of autophagy inhibition. This was evident from the elevated presence of primary autophagy-related proteins (LC3I/II, Beclin1, and p62) in aged MSCs when compared to their younger counterparts (Figure 3a,b–d). Subsequently, we embarked on an investigation to determine whether the suppression of miR-873-5p could alleviate MSC senescence by stimulating autophagy. Suppression of miR-873-5p in aged MSCs triggered elevated manifestation of p62, LC3I/II, and Beclin1, indicating that it effectively activated autophagy in these cells. Moreover, the decreased expression of p53 and p21 in aged MSCs treated with miR-873-5p suppression relative to untreated aged MSCs suggested that miR-873-5p inhibition rejuvenated aged MSCs through autophagy induction (Figure 3a,f,g). Furthermore, our previous research underscored the significance of the AMPK signaling pathway in the regulation of autophagy. In light of this, we sought to explore whether miR-873-5p supervised autophagy via the AMPK signaling pathway. Interestingly, it was observed that the suppression of miR-873-5p led to the stimulation of AMPK phosphorylation (Figure 3a,e). To further validate this conclusion, we employed both miR-873-5p and the autophagy inhibitor 3-methyladenine (3-MA) in aged MSCs. Treatment with 3-MA decreased autophagy and resulted in elevated levels of p53 and p21. These outcomes collectively suggest that the downregulation of miR-873-5p rejuvenates aging MSCs by promoting autophagy (Figure 3a). In brief, our investigation demonstrates that miR-873-5p suppression can counteract MSC aging by enhancing autophagy, with the AMPK signaling pathway playing a pivotal role in this process.

Figure 3.

miR-873-5p inhibition rejuvenates aged MSCs via inducing autophagy. (A–G) Western blotting was performed to assess the protein expression of LC3II/I, Beclin1, p62, AMPK, p-AMPK, p53, and p21 in aged MSCs transfected with miR-control, miR-873-5p inhibitor, or miR-873-5p inhibitor + 3-MA. The quantification of these protein levels was carried out. Continuous variables and categorical variables are reported as means ± SEM and percentages. n = 3. ***p < 0.001.

Cellular Senescence in MSCs is Regulated by miR-873-5p Via Targeting of Cab39

TargetScan (http://www.targetscan.org/) was utilized to reveal the expected target genes of miR-873-5p, and it unveiled a presumed binding site within the 3′UTR of calcium-binding protein 39 (Cab39) (Figure 4a). Cab39 has the capacity to modulate LKB1 activity and initiate AMPK phosphorylation as a constituent of the trimeric liver kinase B1 (LKB1)-STRAD-Cab39 complex. The Western blotting results from our study unequivocally demonstrate the effect of miR-873-5p on Cab39 expression. Specifically, the introduction of miR-873-5p mimics led to a substantial suppression of Cab39 expression (Figure 4b,c). Conversely, when we utilized the miR-873-5p inhibitor in AMSCs, it resulted in an elevation of Cab39 expression, signifying a clear negative correlation between the two genes (Figure 4d,e). To further elucidate the molecular mechanism, we employed a dual-luciferase reporter gene assay. The assay provided unambiguous evidence that miR-873-5p mimics significantly inhibited the Cab39 wild-type (WT) reporter’s luciferase output, without impacting the luciferase levels of mutant-type (MUT) Cab39 (Figure 4f). These data confirm that miR-873-5p directly targets Cab39. In summary, our findings strongly indicate that the regulation of cellular senescence in MSCs is governed by miR-873-5p, which exerts its effect by targeting Cab39.

Figure 4.

miR-873-5p controls MSC cellular senescence by targeting Cab39. (A) Potential binding sites for miR-873-5p were identified within the 3′UTR of Cab39. (B) 293T cells were cotransfected with either a miR-873-5p mimic or miRNA control, along with a luciferase reporter vector containing the wild-type (WT) or mutant 3′UTR of Cab39. (C) A Western blot assay was conducted to quantitatively assess Cab39 levels in YMSCs after transfection with a scrambled miRNA control, miR-873-5p mimic, or miR-873-5p inhibitor. Continuous variables and categorical variables are expressed as means ± SEM and percentages. n = 3. **p < 0.01, ***p < 0.001.

Anti-miR-873-5p-AMSCs Transplantation in a Rat Model of MI-Protected Cardiac Function

In this study, we conducted experiments involving the administration of anti-miR-873-5p-AMSCs into infarcted rat hearts to investigate whether the curtailment of miR-873-5p in AMSCs could enhance their therapeutic effects. We assessed the outcomes 28 days after inducing MI using representative M-mode echocardiographic images (Figure 5a). At 28 days post-MI, it was evident that in comparison to the MI group, both LVEF and LVFS saw crucial enhancements in all MSC-transplanted groups. Interestingly, the LVEF and LVFS were notably reduced in the AMSC group, in contrast to the YMSC group, indicating a decline in cardiac function. However, in the anti-873-5p-AMSC group, there was partial recovery, suggesting that anti-873-5p-AMSCs were more effective than AMSCs in restoring cardiac function after MI (Figure 5a,d,e). Furthermore, we assessed the infarct size at 28 days after myocardial infarction using Masson’s three-color staining. In the AMSC group, the infarct size was notably greater than that in the YMSC group (Figure 5b,e). However, in the anti-873-5p-AMSC group, the infarct size was notably smaller than that in the AMSC group, indicating enhanced cardioprotection (Figure 5b,e). Moreover, we evaluated inflammatory cell infiltration into cardiac tissue. In all MSC-transplanted groups, there was a substantial reduction in inflammatory cell infiltration compared with the MI group, and the anti-miR-873-5p-AMSC group exhibited even less infiltration when compared with the AMSC group (Figure 5f). Taken together, these extensive results indicate that blocking miR-873-5p in AMSCs can enhance cardioprotection in the rat model of AMI.

Figure 5.

Anti-miR-873-5p-AMSCs transplantation in a rat model of MI-protected cardiac function. (A) M-mode echocardiography images were captured 28 days after myocardial infarction (MI) in various experimental groups. (B) Masson’s trichrome staining images and the quantitative analysis of infarction size were performed in control mice or aged mice treated with PBS, YMSCs, AMSCs, or anti-miR-873-5p-AMSCs. Scale bar = 2 mm. (C) Hematoxylin and eosin (HE) staining was conducted at the border zone 28 days after MI. (D,E) Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured using echocardiography 28 days after MI. Scale bar = 2 mm. (F) Quantitative analysis of heart fibrosis was carried out among different experimental groups. Continuous variables and categorical variables are presented as means ± SEM and percentages, respectively. n = 5 animals for each group. *P < 0.05, **P < 0.01, ****P < 0.0001.

Transplantation of Anti-miR-873-5p-AMSC Promotes Angiogenesis in Infarcted Hearts

The staining for human nuclear antigen (HNA) clearly indicated that the survival of MSCs was markedly enhanced in the anti-miR-873-5p-AMSC group, in contrast to the AMSC group. Notably, the YMSC group showed the most abundant surviving MSCs in heart tissue. In Figure 6a,b, we observe that HNA expression in the anti-miR-873-5p-AMSC group exhibited a significant improvement compared to the AMSC group. To assess the neovascularization effect of MSC grafting, arteriolar density and microvessel density in mouse hearts were evaluated using SMA staining and CD31 staining 28 days after the procedure. Notably, all MSC-treated groups exhibited a substantial increase in arteriolar density when compared to the MI group, as illustrated in Figure 6c. Among these groups, the YMSC group displayed the highest arteriolar density. Additionally, the anti-miR-873-5p-AMSC group showed a higher number of arterioles compared to that of the AMSC group (Figure 6d). When it comes to capillary densities in the various MSC-treated groups, our findings remained consistent. The YMSC group demonstrated the highest capillary density in heart tissue, while the anti-miR-873-5p-AMSC group exhibited a boosted capillary count compared to the AMSC group, as depicted in Figure 6f.

Figure 6.

Transplantation of anti-miR-873-5p-AMSC promotes angiogenesis in infarcted hearts. (A,B) HNA staining images and the quantitative analysis of cell survival were performed in aged mice 28 days after MI and treated with YMSCs, AMSCs, or anti-miR-873-5p-AMSCs. Scale bar = 50 μm. (C) Images of α-SMA staining of heart tissue from control or aged mice with MI, treated with PBS, YMSCs, AMSCs, or anti-miR-873-5p-AMSCs. Scale bar = 200 μm. (D) Quantitative analysis of arteriole density in the different treatment groups. (E) Images of CD31 staining of heart tissue from control or aged MI mice treated with PBS, YMSCs, AMSCs, or anti-miR-873-5p-AMSCs. Scale bar = 50 μm. (F) Quantitative assessment of capillary density among the various treatment groups. Data are presented as the mean ± SEM n = 5. **p < 0.01, ****p < 0.0001.

Transplantation of Anti-miR-873-5p-AMSCs Reduces Cardiomyocyte Mortality and Increases Con43 Expression in Infarcted Rat Hearts

To gauge the apoptosis-inhibiting effects of MSC transplantation, we evaluated cardiomyocyte apoptosis within the ischemic region using TUNEL and cleaved caspase-3 staining, as shown in Figure 7a,b. Remarkably, all groups that received MSC transplantation exhibited a notable decrease in the rate of cardiomyocyte apoptosis in relation to the MI group. Among these groups, the YMSC group displayed the lowest level of apoptosis. Furthermore, hearts in the anti-miR-873-5p-AMSC group demonstrated fewer apoptotic cardiomyocytes compared to the AMSC group, as depicted in Figure 7c,d. In Figure 7e, we noted a remarkable rise in the presence of Cx43, the predominant gap junction protein in the heart, in all MSC-treated groups when contrasted with the MI group. Additionally, the anti-miR-873-5p-AMSC group exhibited higher Cx43 expression levels compared to those of the AMSC group. Based on our findings, it can be concluded that anti-miR-873-5p-AMSC grafting effectively reduces Cx43 expression and cardiomyocyte mortality in rat hearts following myocardial infarction.

Figure 7.

Anti-miR-873-5p-AMSC transplantation decreases cardiomyocyte death and improves Con43 expression in mice hearts with infarction. (A) Images of TUNEL staining of heart tissue from mice with MI treated with injections of PBS, YMSCs, AMSCs, or anti-miR-873-5p-AMSCs. Scale bar = 50 μm. (B) Quantitative analysis of cardiomyocyte apoptosis in the various treatment groups. (C) Cleaved caspase-3 staining images at the border zone 28 days after MI. Scale bar = 50 μm. (D) Quantitative assessment of cleaved-caspase-3+ cells at the border zone across different treatment groups. (E) Border zone sections collected 4 weeks post-MI were subjected to immunofluorescent staining for Connexin 43 (Con43) expression. Red represents Connexin 43; green depicts α-actin; blue represents DAPI-stained nuclei. Scale bar = 50 μm. Continuous variables are expressed as means ± SEM n = 5. ***p < 0.001, ****p < 0.0001.

Discussion

In this study, a number of significant discoveries have been reported. First, it identified a novel observation that miR-873-5p is considerably heightened in AMSCs and may play a role in promoting the progressive aging of MSCs. Second, the study revealed that miR-873-5p exerts its regulatory influence on MSC senescence by modulating autophagy through a mechanism involving Cab39 and AMPK. Furthermore, the suppression of miR-873-5p was shown to have a beneficial impact on AMSCs, enhancing their cardioprotective properties, rejuvenating them, supporting their survival, and promoting angiogenesis in rat hearts that had suffered from infarction.

In recent years, MSC transplantation has emerged as a promising approach for myocardial infarction (MI) restoration in both animal studies and early human tests owing to factors such as their accessibility, varied origins, pluripotency, and immune-shielded status.²⁶⁻²⁸ However, while allogeneic MSC transplantation offers immediate benefits for MI, its long-term efficacy is limited compared to that of syngeneic MSCs because of reduced cell survival due to immune rejection. Autologous MSCs have the potential to address this limitation and strengthen the safeguarding abilities of the cells. Nevertheless, native MSCs derived from elderly donors exhibit lower homeostatic and regenerative abilities due to senescence. Studies involving the transplantation of autologous MSCs after MI in rats have shown reduced potential for cardiac repair compared with younger donor-derived MSCs (YMSCs). Our investigation corroborates these findings, demonstrating that MSCs from elderly donors exhibit lower proliferation and differentiation abilities, enhanced SA-β-gal performance, and diminished paracrine activities, all indicative of senescence. Rejuvenating aging MSCs to enhance their positive therapeutic outcomes in cardiovascular disorders has garnered significant interest. Various cutting-edge methods, including gene alteration and pharmacological pretreatment, have been explored for this purpose. Nevertheless, the precise processes responsible for MSC aging remain unclear.

Recent research has identified several miRNAs that control MSC senescence through different pathways.²⁹⁻³² For instance, miR-195 overexpression can trigger cell aging in MSCs by suppressing telomerase reverse transcriptase, reducing their regenerative capacity.³³ Conversely, elimination of miR-195 can slow down MSC deterioration and boost cardiac healing in postinfarction mice. Diminishment of miR-543 and miR-590-3p impairs MSCs’ ability to differentiate into adipogenic and clonogenic cells, leading to the senescence phenotype.³⁴ Elevated levels of miR-335 in MSCs induces cellular aging, as indicated by heightened SA-β-gal function, elevated p16 protein levels, reduced cell multiplication, and compromised immunoregulatory and maturation potential.³⁵ Our prior research has demonstrated that blocking miR-873-5p yields cardioprotective effects by promoting AMPK activation in cardiomyocytes via XIAP targeting.¹⁵ Additionally, our current study reveals significantly elevated levels of miR-873-5p in human adipose-derived MSCs (AMSCs) and serum from aged donors. This observation suggests that miR-873-5p may indeed play a crucial role in controlling the aging of MSCs. We have also discovered that elevated levels of miR-873-5p in YMSCs can ameliorate the aging cellular profile by enhancing SA-β-gal work, elevating the levels of P21 and P53, and reducing the number of Ki67-positive cells. Conversely, the suppression of miR-873-5p in AMSCs reduces SA-β-gal operation and promotes angiogenesis and cell multiplication. In our mouse model, the transplantation of anti-miR-873-5p-AMSCs proved to be more effective than the transplantation of AMSCs alone in reducing cardiac remodeling and improving cardiac function following infarction. These results underscore the potential of miR-873-5p inhibition for MSC regeneration, while miR-873-5p itself can hasten senescence in these cells. Nevertheless, the specific mechanism by which miR-873-5p governs MSC senescence remains elusive.

The antiaging benefits of autophagy have gained increasing recognition in recent years. The hypoxia-inducing factor 1α/AIMP3 signaling pathway has been substantiated as an activator of autophagy, effectively retarding the aging process in MSCs.³⁶ Conversely, the autophagy inhibitor 3-MA, as opposed to the autophagy activator rapamycin, has been shown to expedite the aging of immature MSCs. Inducing autophagy presents a promising avenue for rejuvenating the MSC function. Our investigations have unveiled that suppressing miR-873-5p leads to an increase in autophagy levels compared to aged MSCs, and these effects were partially reversed by 3-MA. This suggests that miR-873-5p acts as a key contributor to driving MSC aging through the regulation of autophagy.

Emerging evidence suggests that AMPK signaling is a key regulator of autophagy. However, it remains uncertain whether miR-873-5p exerts control over the AMPK signaling pathway, thereby influencing autophagy. In our quest to identify miR-873-5p targets, we pinpointed Cab39 as a gene specifically regulated by miR-873-5p. Cab39 is an upstream coactivator of AMPK and is known to possess cell-protective properties. Our study has revealed a marked reduction in the manifestation of Cab39 and p-AMPK in AMSCs in contrast to YMSCs, indicating a direct link between Cab39/AMPK signaling and the cellular aging of MSCs. Furthermore, the suppression of miR-873-5p generated increased quantities of Cab39 and p-AMPK expression. We delved into the possibility that miR-873-5p inhibitor-induced autophagy might be mediated through the Cab39/AMPK signaling pathway. Nevertheless, it is important to highlight that AMPK has diverse alternative signaling mechanisms governing its activity. Consequently, we cannot dismiss the possibility that miR-873-5p may employ other molecules or pathways beyond Cab39 to modulate the AMPK activity. Further research is needed to unravel the intricate mechanisms by which miR-873-5p influences MSC aging and autophagy regulation.

Our study also presents certain limitations. First, we exclusively investigated miR-873-5p in AMSCs. Further research is imperative to elucidate the functions of other abundant miRNAs within AMSCs. Second, it remains uncertain whether miR-873-5p controls any other targets aside from Cab39 that contribute to MSCs’ senescence. Third, the duration of anti-miR-873-5p-AMSC’s ability to enhance heart function postinfarction remains unknown. Lastly, independent and rigorous data, including molecular profiling, are essential for verifying our discoveries.

In summary, our results highlight that suppressing miR-873-5p rejuvenates aging MSCs by modulating autophagy, offering a promising avenue to enhance MSC-mediated cardioprotection in aging hearts following infarction. The primary driver of this revitalization is the Cab39-AMPK signaling pathway.

Data Availability Statement

Most of the data sets supporting the conclusions of this article are included within this article and the additional files. The data sets used or analyzed during the current study are available on reasonable request.

Author Contributions

^⊥ W.Z., W.D., and R.D. contributed equally to this study. W.W.Z., W.D., Y.M.H., and B.H. contributed to the design of the study. W.W.Z., W.D., R.D., Y.L., B.Z., X.J., Z.S.D., W.H., and S.S. performed the experiments. W.W.Z., W.D., R.H.W., Y.M.H., and B.H. contributed to the writing of the manuscript. W.W.Z., W.D., and H.R.W. contributed to the material support of the study. All authors read and approved the final manuscript.

This study was supported by grants from the Youth medical innovation project of Xuzhou Health Commission (XWKYSL20210231, XWKYHT20230082), Xuzhou Medical University Outstanding Talent Fund (XYFY202333, XYFY202336), the NSFC Incubation Program of GDPH (KY012021167 to Y.H.), and the Natural Science Foundation of Jiangsu Province (BK20220472).

Ethics approval and consent to participate: animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Xuzhou Medical University.

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Pharmacology & Translational Sciencevirtual special issue “Nucleosides, Nucleotides, and Nucleic Acids as Therapeutics”.