Sarcopenia-derived exosomal micro-RNA 16-5p disturbs cardio-repair via a pro-apoptotic mechanism in myocardial infarction in mice

Sarcopenia is a pathophysiological malfunction induced by skeletal muscle atrophy. Several studies reported an association between sarcopenia-induced cardiac cachexia and poor prognosis in heart disease. However, due to lack of an established animal models, the underlying mechanism of disturbed cardiac repair accompanied with sarcopenia remains poorly understood. Here, we developed a novel sarcopenia-induced cardiac repair disturbance mouse model induced by tail suspension (TS) after cardiac ischemia and reperfusion (I/R). Importantly, we identified a specific exosomal-microRNA marker, miR-16-5p, in the circulating exosomes of I/R-TS mice. Of note, sarcopenia after I/R disturbed cardiac repair and raised the level of circulating-exosomal-miR-16-5p secreting from both the atrophic limbs and heart of TS mice. Likewise, miR-16-5p mimic plasmid disturbed cardiac repair in I/R mice directly. Additionally, in neonatal rat ventricular myocytes (NRVMs) cultured in vitro under hypoxic conditions in the presence of a miR-16-5p mimic, we observed increased apoptosis through p53 and Caspase3 upregulation, and also clarified that autophagosomes were decreased in NRVMs via SESN1 transcript interference-mediated mTOR activation. In conclusion, we show the pro-apoptotic effect of sarcopenia-derived miR-16-5p, which may be behind the exacerbation of myocardial infarction. Therefore, miR-16-5p can be a novel therapeutic target in the context of cardiac repair disturbances in sarcopenia–cachexia.


Skeletal muscle atrophy in I/R mice is induced via sustained tail-suspension (TS).
Experimental sarcopenia was induced in I/R mice using the modified Morey's tail-suspension (TS) model from days 1 to 8 after I/R (Fig. 1a) 22 . To assess the effect of modified tail-suspension on the skeletal muscles, I/R mice subjected or not to TS [TS (+), or TS (−), respectively] were randomly assigned to two groups and compared. One week after were significantly decreased in TS (+) mice compared to in TS (−) mice (Fig. 1b, c). Additionally, as per the histological analysis, the gastrocnemius muscle of TS (+) mice showed atrophied muscle fibers and increased interstitial tissues with localized inflammatory cell infiltration (Fig. 1d). Of note, comparing the fiber crosssectional area (CSA) present per a unit area in the two groups revealed an evident reduction in TS (+) mice compared to TS (−) mice [TS (−) vs. TS (+); 1421.1 ± 81.4 vs. 1193.4 ± 63 0.7 µm 2 , respectively; p = 0.030; Fig. 1e, f]. In line with these results, the liver and lungs weight of TS (+) mice were slightly decreased after modified tail-suspension compared to those in TS (−) mice, and the body weight was comparable between the two groups (Supplementary Figure 1).
We also evaluated the infarct size in I/R-mice using Masson's Trichrome staining at day 29 to clarify the effect of the modified TS method on persistent myocardial damage. An area of intense staining in the myocardium of I/R-TS (+) mice was larger emphasized than that of I/R-TS (−) mice (Fig. 2d). Further, the infarct size in I/R-TS (+) mice was significantly greater than that of I/R-TS (−) mice (16.3 ± 4.2 vs. 6.3 ± 1.9%, respectively; p = 0.041, Fig. 2e).  www.nature.com/scientificreports/ Exosomal micro-RNAs in the context of experimental sarcopenia after I/R. Next, circulating exosomes were extracted from the whole blood of both I/R mice after the release of TS (day 8) and the total RNA of exosomes was purified and subjected to micro-RNA array analysis; 3D-Gene global miRNA microarray mouse chips encompassing all mouse miRNAs available on the Sanger miRBase were used in the two groups (n = 3 mice per group) to identify a specific exosomal miRNA which exerted the cardio-repair disturbance in I/R-TS (+) mice. A comprehensive cluster analysis of the expression of miRNAs in the exosomes from both groups of I/R mice showed that the cardio-repair disturbance was associated with the differential expression of 68 miRNAs [fold change > ± 2.0 (log2 > ± 1.0), Fig. 3a].
Notably, the expression of 42 miRNAs (among the 68 differentially expressed) was significantly up or down regulated in I/R-TS (+) mice (p < 0.05, Supplementary Table 1). Further, we identified two upregulated candidate micro-RNAs, miR-16-5-p and miR-144-3p, showing with a > 4-fold change (log 2 > 2.0) as shown in the Volcano plot analysis (Fig. 3b). Finally, we selected these two micro-RNA candidates (miR-16-5-p, miR-144-3p), and miR-24-3p (well known as cancer and I/R heart related miRNA, which had a high expression ratio but no statistical variance in this microarray analysis; validation control) for subsequent validation via qRT-PCR. The expression level of miR-16-5p and miR-144-3p was not significantly different between mouse with and without I/R condition, however, we confirmed that the expression of exosomal miR-16-5p was clearly upregulated in response to the tail-suspension after I/R in this validation study [expression ratio; I/R (+)-TS (−) vs. I/R (+)-TS (+) = 1.0 ± 0.6 vs. 9.7 ± 4.5%, respectively; p = 0.045, Fig. 3c]. Based on our results, we selected a miR-16-5p as the candidate miRNA most likely associated with the cardio-repair disturbance in I/R mice with sarcopenia.
Circulating miR-16-5p is derived from the atrophic limbs and heart of sarcopenic mice and directly interferes with the restoration of LV dysfunction in I/R mice. As a result of the miR-16-5p mimic in vitro study, an exosomal miR-16-5p might affect the cardio-repair disturbance due to induce the pro-apoptotic effect to ischemic cardiomyocytes in sarcopenia mice after I/R. Accordingly, the following question was raised; from which tissue did the exosomal miR-16-5p original from in mice? To address this query, we performed organ profiling using qRT-PCR to assess the exosomal miR-16-5p source in the context of our in vivo model by determining the expression of miR-16-5p in different tissues (brain, heart, limb, liver, lung, aorta, pancreas, stomach, bone marrow, kidney, and prostate) of sarcopenic mice without I/R induction (Fig. 6a). In general, the TS method is recognized as a model of depression, however, no difference in the expression of miR-16-5p was observed in the brain of sarcopenic mice. Similarly, no difference in the expression levels of miR-16-5p was also observed in the other tissues (liver, lung, aorta, pancreas, stomach, kidney, prostate) of sarcopenic mice in the two groups, in contrast, the expression of miR-16-5p in the bone marrow of TS (+) mice was decreased versus that in TS (−) mice (0.28 ± 0.05 vs. 1.00 ± 0.26, respectively; p = 0.0367). Meanwhile, the expression of miR-16-5p in the atrophic limbs of TS (+) mice 7 days after a tail-suspension was significantly increased versus that in TS (−) mice (1.0 ± 0.12 vs. 1.53 ± 0.15, respectively; p = 0.0353). In addition, the expression of miR-16-5p in the heart of TS (+) mice was also significantly increased versus that in TS (−) mice, even in the absence of I/R (1.02 ± 0.13 vs. 2.21 ± 0.30, respectively; p = 0.0137).

Discussion
Here, we developed a robust I/R-based cardiac impairment mice model in the presence of sarcopenia. We, further, successfully employed our novel model to identify a cardiotoxic exosomal micro-RNA and characterized miR-16-5p, which acts by decreasing autophagy and promoting cardiomyocyte apoptosis, as a pivotal player in cardiac impairment. The prevention of sarcopenia after myocardial infarction remains a global health care issue, therefore, we should not also let it forgetting as a pathophysiological cardio-repair intervention against an exosomal micro-RNA interference. Micro-RNAs are known interference regulators of gene transcription, and play an important role in the autocrine and/or paracrine repair of injured tissues. For instance, several micro-RNAs, including miR-21 25 , miR-29 26 , and miR-25 27 , were defined as biomarkers in the context of cardiac hypertrophy, MI, and HF 28 . Additionally, MiR-1 29 , miR-133 30 , and miR-208 31 were reported to be upregulated in the context of embryonic heart development from cardiac crescent to fetal heart as autocrine regulators 32 . These micro-RNAs, known as cardiac regulators, have been mainly studied concerning their associations with the cardiac cycle, regeneration, and cardiomyocyte proliferation 32 . However, much less is known in the context of pathophysiological conditions such as HF and/or cardiac cachexia. Of note, a few "cardio-regulated" micro-RNAs suggested as biomarkers of HF may be mobilized from other organs in patients with heart disease. In the present study, we established a cardio-repair disturbance model mimicking cardiac cachexia based on limb unloading-induced sarcopenia after myocardial ischemia/reperfusion. Previously, Hughes et al. reported that miR-31 in the atrophic skeletal muscles of aged rats was transiently upregulated after mechanical limb unloading, injuring the skeletal muscles in an autocrine manner 33 . Here, via multiorgan profiling in sarcopenic mice we suggested that miR-16-5p upregulation in the atrophic limbs was streamed as circulating-exosomal micro-RNAs, which reached and subsequently impacted the heart. Based on micro-RNA mimic in vitro studies, sarcopenia-induced circulating-exosomal miR-16-5p may be closely associated with deterioration of an injured heart and generate a state of "cardiac cachexia". Interestingly, although there were no apparent increased preload signs in the lungs and liver of sarcopenic mice miR-16-5p was also elevated in the heart after limb unloading without I/R. Probably, the induction of sarcopenia not only leads to skeletal muscle atrophy, to a prodromal state of "cardiac cachexia" like with downregulated miR-16-5p expression in bone marrow, which may, in turn, also upregulate cardiac miR-16-5p expression. Taken together, our results collectively suggest that miR-16-5p is a novel "cardio-regulated" micro-RNA whose expression is induced in the context of sarcopenia and is responsible for "cardiac cachexia" (Fig. 6d). miR-16 belongs to the micro-RNA-15 family (consisting of miR-15a/b, miR-16-1/2, miR-195 and miR-497), and is a well-known tumor suppressor, highly expressed in several cancers, including prostate cancer, lung cancer, and chronic lymphocytic lymphoma. Additionally, it was previously reported that the members of the micro-RNA-15 family are important for the regulation of the differentiation of cardiomyocytes and skeletal muscle cells. Porrello et al. described that the inhibition of the expression of miR-195 at an early postnatal stage decreased the proliferation of myocytes not only in embryo-but also in postnatal hearts and exacerbated to the left ventricular systolic function in adult mice after MI 34 . miR-16-5p, the one this study focuses on has the homologous sequence "AGC AGC ", found in all of the micro-RNA-15 family members, and regulates the transcription of genes related to cell proliferation, regeneration, and death. Recently, Cai et al. reported that miR-16-5p directly targets the SESN1 gene impacting the proliferation and apoptosis of myoblasts, and consequently the differentiation of skeletal muscles 35 . Additionally, Li et al. described the role of SESN1/2 in doxorubicin cardiotoxicity using SESN1/2 double-knockout mice 36 ; however, the regulator of SESN1 in the context of cardiac injury has not yet been determined in their model. In the current study, when we transfected NRVMs with a miR-16-5p mimic-encoding plasmid, a pro-apoptotic effect was observed under hypoxic conditions in vitro; of note, the same was not true under normoxic conditions. In general, excessive oxidative stress leads to apoptosis through increased p53 expression; this said, p53 may also stimulate cytoprotective pathway to maintain cell homeostasis, regulating autophagy by mTOR dephosphorylation, via SESN1 upregulation 37 . Because miR-16-5p directly inhibits the transcription of SESN1, miR-16-5p has a pro-apoptotic effect, leading to an imbalance of apoptosis and autophagy under oxidative stress (Fig. 6d). Interestingly, in the current study, miR-16-5p could not promote the apoptosis of NRVMs under normoxic conditions, and the cardio-repair disturbance in the context of the I/R mouse model used was not observed in the absence of sarcopenia. This suggests that exosomal miR-16-5p, in the absence of oxidative stress, does not impact apoptosis and autophagy in cardiomyocytes; only exosomal miR-16-5p accompanied with a sarcopenia-cachexia intervention promotes the deterioration of myocardial injury via a pathophysiological mal-adaptation. As well known in previous study, autophagy usually plays a pivotal role in controlling cell viability, providing the necessary nutrients during starvation. Therefore, the mobilization of exosomal miR-16-5p may inhibit the autophagy-based self-repair of injured organs in a sarcopenia-cachexia environment. That is one reason explaining why a conventional clinical approach to treat myocardial ischemia would not revert cardio-repair disturbances after MI in the context of sarcopenia. Hence whenever early adaptation interventions (e.g., exercise intervention) after MI are impossible, due to aging or severe HF, therapeutic approaches targeting the miR-16-5p-SESN1 axis may be ideal alternatives, in the future.
Certain limitations were noted in the current study. For instance, in this study, we did not adopt a genetic model mouse to validate the loss of function of miR- 16- Figure S2) after I/R without tail suspension. Meanwhile, TS (+) mouse without I/R had no heart failure and less alteration of general condition by limb unloading (Supplementary Figure S1). Therefore, after myocardial ischemia, a sarcopenia induced by limb unloading may lead to a constitutional specific alteration as a "cardiac cachexia", which associated with circulating exosomal-miR-16-5p acting as an "external" secretory cardiac regulated micro RNA-as well as cardiac endogenous miR-16-5p. Direct interactions between limb unloading and endogenous cardiac miR-16-5p will be investigated further in subsequent studies.
In conclusion, we show that the induction of sarcopenia after I/R injury promotes cardiac repair disturbance together with the increased expression of circulating-exosomal-miR-16-5p. We further demonstrate that miR-16-5p promotes apoptosis in cardiomyocytes via SESN1, hence, the miR-16-5p-SESN1 axis should be considered as a potential target for new treatment strategies for heart disease patients that are unable to undergo exercise intervention-based early adaptation due to aging or severe HF. Mouse model of I/R, sarcopenia, and miR mimic plasmid injection. 8-10-week-old C57BL/6 male mice were anesthetized and ventilated with 3% isoflurane after intubation. The left anterior (coronary) descending artery (LAD) was occluded directly under the left atrium using monofilament nylon 8-0 sutures (Ethicon, Somerville, NJ, USA) for 45 min; then, the occlusion was released (I/R procedure). The occlusion time of the LAD was designed as 45 min to enable for the partial recovery of the contraction of the left ventricle. Additionally, for sarcopenia induction, mice were tail-suspended for 7 days in individual cages. This TS protocol employs a pulley block that maintains forelimb activity and does not interfere with food or fluid intake. Moreover, a horizontal balance must be achieved to avoid applying a steep angle to the animal's body, thus, permitting normal weight bearing on the forelimbs, as has been described previously 39,40 . I/R mice were injected with miR-16-5p mimic plasmid (sense-5ʹ-uagcagcacguaaauauuggcg-3ʹ, antisense-5ʹ-cgccaauauuuacgugcugcuauu-3ʹ) or negative control-miR mimic plasmid (sense-5ʹ-auccgcgcgauaguacguaTT-3ʹ, antisense-5ʹ-uacguacuaucgcgcggauTT-3ʹ) (Koken CO, Tokyo, Japan) at day 1 and 4 after coronary reperfusion. In brief, 4 nmol/body of miR-16-5p mimic, or control-miR mimic was mixed with 100 µL Dulbecco's Phosphate Buffered Saline (DPBS). Atelocollagen (AteloGene ® , Koken CO) was diluted in an equal volume of DPBS to attain a final concentration of 0.1% by pipetting up and down for 20 times, and rotating for 15 min at 4 °C. After these two solutions were mixed together by pipetting up and down for 20 times, the mixture (200 μL for each mouse) was then delivered into each mice via tail vein with an insulin syringe (27G, 1 mL). LV function of I/R mice was evaluated at day 7 after miR-mimic plasmid injection by echocardiography.

Histological analysis (hematoxylin and eosin, and Masson's trichrome staining).
Gastrocnemius muscle samples were harvested from TS mice (n = 10, respectively) 8 days after tail-suspension. Hearts were harvested from TS mice (n = 6, respectively) 21 days after tail-suspension. All samples were fixed in 4%  www.nature.com/scientificreports/ paraformaldehyde (PFA), followed by treatment with sucrose solution. Frozen sections (7 µm for the hearts and 10 µm for skeletal muscles) were then obtained. Three gastrocnemius muscle samples [TS (+) = 2, TS (−) = 1] that were unsuitable for analysis due to damage during storage or freezing were treated as outliers. Hematoxylin and eosin staining 38 were conducted on gastrocnemius sections to analyze the myofiber size. Images were acquired using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan), and the area of myofibers was measured using the BZ-X Analyzer software (BZ-H3A/H3C ver.1.3.1.1 Keyence. Co.). The heart sections were subjected to Masson's Trichrome staining 38 to evaluate fibrosis; the fibrotic area was measured using the Image-J software (ver.1.53a; National Institutes of Health, Bethesda, MD, USA. http:// imagej. nih. gov/ ij). Additionally, the infarct size was calculated as the percentage of fibrosis area within the total LV area.
Echocardiography. 30 (short term) and 12 (long term) mice were divided into groups using a random number table after the surgery and transthoracic echocardiography was performed to evaluate heart function before and 1, 8, and 29 days after the I/R procedure, using the Vevo 660 system (VisualSonics, Toronto, Canada). B-mode images of hearts were recorded from the parasternal short and long-axis view. The end-systolic and enddiastolic left intraventricular areas (basal, mid and apical) in the short-axis view were measured. The LV ejection fraction (LVEF) was calculated using the following formula: V = (area mid-ventricular + area apical + area basal) × h/3, where h = ventricular length. The ejection fraction was calculated for both methods using the formula: EF = (EDV − ESV)/EDV × 100. The assessment was performed in a blinded manner.
Extraction of micro-RNAs from circulating-exosomes. Total exosomes were isolated from the serum using the Total Exosome Isolation Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the collected blood was centrifuged at 2000g for 10 min to obtain the serum. The serum was further centrifuged at 2000g for 30 min to completely remove the cells and debris, and 40 µL of reagent was added to 200 µL of the obtained serum and incubated at 4 °C for 30 min. After incubation, samples were centrifuged at 1000g for 10 min, and the exosomes remaining at the bottom of the tube were lysed in a resuspension buffer. Total RNA www.nature.com/scientificreports/ was extracted from the resulting exosome-containing samples using the Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific) and reversely-transcribed into cDNA using the TaqMan Micro-RNA Reverse Transcription Kit, as per the manufacturers' instructions.
miRNA microarray analysis. For the micro-RNA microarray analysis, total RNA samples were extracted from circulating-exosome of TS (−) and TS (+) mice (n = 3), and their quality was checked using the Bioanalyzer system (Agilent, Santa Clara, CA, USA). miRNA expression was analyzed using the 3D-Gene miRNA Oligo chip and 3D-Gene miRNA labeling kit. Briefly, half volumes of labeled RNAs were hybridized onto a 3D-Gene miRNA Oligo chip designed to detect 2565 miRNA sequences; the annotation and oligonucleotide sequences of the probes were conformed to the miRbase. Hybridization signals were scanned using the 3D-Gene Scanner3000 and processed using the 3D-Gene Extraction software (All materials; Toray, Tokyo, Japan). The detected signals for each gene were normalized using the global normalization method. The candidate micro-RNAs assigned as differentially expressed with an adjusted p-value < 0.05 (one-sided t test) were narrowed down among genes differentially expressed over four-fold between the 2 groups.  microRNA mimic transfection in the context of NRVMs under hypoxic conditions. NRVMs were obtained from neonatal (1-day old) SD rat hearts following the manufacturer's protocol 38 . NRVMs were allowed to reach 50% confluency, to determine the conditions that enable the best visualization of NRVM apoptosis and autophagosomes. Six days after seeding to allow NRVM maturation, the culture media was replaced with serum-free medium (DMEM/F12, Thermo Fisher Scientific K.K. Japan), and then, cells were cultured for 2 days in a hypoxic environment containing 5% CO 2 , 1% O 2 , and 94% NO 2 in an incubator. To investigate the effect of miR-16-5p in the context of NRVMs, a miR-16-5p mimic [50 nM; miRVana miRNA mimics (Ambion)] was transfected using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) for 2 days in a hypoxic environment. The TUNEL assay and the detection of autophagosomes were performed after hypoxic cultivation for 2 days.

Quantitative RT-PCR (qRT-PCR
TUNEL analysis. For the terminal deoxynucleotidyl TUNEL assay, cells were fixed with 2% paraformaldehyde for 10 min at room temperature. After permeabilization with phosphate-buffered saline containing 0.1% Triton-X and 0.1% sodium citrate for 2 min at 4 °C, cells were incubated with fluorescein isothiocyanate (FITC)conjugated TUNEL reaction mixture (In situ Cell Death Detection kit, Roche Diagnostics, Indianapolis, IN, USA) for 60 min at 37 °C. Samples were then stained with 4ʹ,6-diamidino-2-phenylindole to label the nuclei and visualized under an epifluorescence microscope; the BZ-X Analyzer (Keyence. Co.). TUNEL-positive cells were counted in at least six randomly selected microscopic fields under a 10 × objective.

Detection of autophagosomes. Autophagosomes were detected using the Cell Meter Autophagy
Assay Kit *Green Fluorescence* (AAT Bioquest, Inc., Sunnyvale, CA, USA), after NRVMs were subjected to 1% hypoxia 48 h with or without miR-16-5p mimic transfection. Briefly, the NRVMs were stained with the Autophagy Green™ working solution and incubated at 37 °C for 30 min; then the nuclei were stained with Hoechst 33342 (Lonza, Walkersville, MD, US) for 10 min. Cells were washed three times and examined under a fluorescence microscope, the BZ-X Analyzer. The ratio of autophagosome-positive cells was calculated as per the number of NRVMs containing autophagosomes, versus that of nuclei in at least six randomly selected microscopic fields under a 20 × objective.
Statistical analysis. Experimental data are presented as the mean ± the standard error (SE). The number of samples (n) is disclosed in the respective figure legends. Significance was determined using the Wilcoxon analysis or the student t-test in case of normally distributed. A p-value < 0.05 was considered significant. Data were analyzed using JMP14.0 (JMP, Tokyo, SAS).