MicroRNAs regulating signaling pathways in cardiac fibrosis: potential role of the exercise training

Abstract

Cardiovascular and metabolic diseases such as hypertension, type 2 diabetes, and obesity develop long-term fibrotic processes in the heart, promoting pathological cardiac remodeling, including after myocardial infarction, reparative fibrotic processes also occur. These processes are regulated by many intracellular signaling pathways that have not yet been completely elucidated, including those associated with microRNA (miRNA) expression. miRNAs are small RNA transcripts (18–25 nucleotides in length) that act as posttranscriptionally regulators of gene expression, inhibiting or degrading one or more target messenger RNAs (mRNAs), and proven to be involved in many biological processes such as cell cycle, differentiation, proliferation, migration, and apoptosis, directly affecting the pathophysiology of several diseases, including cardiac fibrosis. Exercise training can modulate the expression of miRNAs and it is known to be beneficial in various cardiovascular diseases, attenuating cardiac fibrosis processes. However, the signaling pathways modulated by the exercise associated with miRNAs in cardiac fibrosis were not fully understood. Thus, this review aims to analyze the expression of miRNAs that modulate signaling pathways in cardiac fibrosis processes that can be regulated by exercise training.

INTRODUCTION

Several cardiac diseases are associated with the cardiac fibrosis (CF) process, such as diabetic cardiomyopathy (1), after myocardial infarction (2), Chagas disease cardiomyopathy (3), hypertension (4), and heart failure (HF; 5). CF is a tissue repair process, characterized by the homeostasis disbalance between the degradation and synthesis of extracellular matrix (ECM) proteins, increasing the expression and deposition of these proteins, inducing fibroblasts concentration and differentiation in the areas of cardiac injury, leading to cardiac remodeling and dysfunction, elevating the possibility of sudden cardiac death and other complications (6, 7).

After an injury, in which occurs the death of some cardiomyocytes, the CF process and the size of fibrotic remodeling depend basically on the extent of the injured area, thus, as cardiomyocytes have negligible regenerative capacity, a cardiac tissue repair program is activated, culminating in the initiation of the CF process (8). This cardiac repair program that promotes CF is regulated by many genes, both protein-coding and noncoding RNAs, including microRNAs (miRNAs; 5). MiRNAs are posttranscriptional regulators of gene expression that control diverse biological processes (9) and actively participate in myocardial remodeling (10). Some strategies have been described to combat the CF process and one of them is exercise training (ExT), such as swimming or running exercise (11), which also modulates the expression of several miRNAs and signaling pathways (12–14). Although ExT is beneficial for individuals with CF, the molecular mechanisms that are modulated by ExT in CF are not completely elucidated. Thus, this review aimed to analyze the miRNAs dysregulated expressed in CF and to compare with the miRNAs regulated by ExT to identify associated signaling pathways and future directions.

MiRNAs ON THE CELL BIOLOGY OF CARDIAC FIBROBLASTS

MiRNAs participate in the regulation of several signaling pathways and cellular processes in practically all cells in the body, and especially in cardiac fibroblasts they also perform important functions. For example, miR-1, miR-133, miR-208, and miR-499 are associated with the process of differentiation of cardiac fibroblasts into cardiomyocytes (15). Another 24 miRNAs were differentially expressed during transforming growth factor-β (TGF-β) induced cardiac fibroblast differentiation. There were three upregulated miRNAs identified: miR-210-3p, miR-325-3p, and miR-325-5p, whereas another 21 miRNAs were downregulated: miR-345-3p, miR-345-5p, miR-483-3p, miR-466b-3p, miR-19b-3p, miR-326-3p, miR-144-3p, miR-19a-3p, miR-129-2-3p, miR-187-3p, miR-429, miR-335, miR-301a-3p, miR-483-5p, miR-1949, miR-b-3p, miR-664-2-5p, miR-190a-5p, miR-879-5p, miR-338-3p, and miR-1843b-3p (16).

Other miRNAs have been associated with the process of cardiac fibroblast proliferation. During this process, miR-29a is poorly expressed and its overexpression has been shown to inhibit the proliferation of these cells (17). In the same sense, miR-1 is also downregulated during the proliferation of cardiac fibroblasts, promoting increased expression of its target genes cyclin D2 and cyclin-dependent kinase 6 (CDK6). When miR-1 was overexpressed and its target genes reduced, the proliferation of cardiac fibroblasts was attenuated (18).

Another miRNA that has reduced expression during the cardiac fibroblast proliferation process is miR-4443. Thus, with decreased expression of miR-4443, it promotes the upregulation of its target gene, thrombospondin-1, with consequent activation of the TGF-β and homologs of the Drosophila protein, mothers against decapentaplegic Mad and the Caenorhabditis elegans protein Sma (SMAD) signaling pathway, generating in addition to cell proliferation, migration, invasion, and differentiation into myofibroblasts with exacerbated collagen production (19).

On the other hand, when miR-1202 is overexpressed, it attenuates the expression of neuronal nitric oxide synthase, inducing an increase in the expression of phosphorylated SMAD-2 and SMAD-3, promoting the synthesis of collagen-1 and collagen-3, favoring the proliferation and differentiation of cardiac fibroblasts (20). In this context of high-expression miRNA, overexpression of miR-214 favors the proliferation of cardiac fibroblasts by inhibiting the expression of its target gene mitofusin-2 and subsequent activation of the ERK 1/2 MAPK pathway (21). In Fig. 1, we summarize the function of miRNAs in these biological processes of cardiac fibroblasts.

Figure 1.

Role of microRNAs (miRNAs) in the biological processes of cardiac fibroblasts.

Thus, it is clear that many miRNAs participate in the natural biological processes of cardiac fibroblasts, as well as being closely related to regulatory processes of cellular differentiation and heart diseases.

MECHANISMS ASSOCIATED TO MYOFIBROBLASTS ACTIVATION IN CARDIAC FIBROSIS

Cardiac fibroblasts account for less than 20% of the nonmyocyte cells in the heart (22), but their function is essential in regulating the phenotype and function of healthy hearts mostly by synthesizing and maintaining the extracellular matrix (ECM) network. They are key regulators of the heart structure and have a major role in cardiac remodeling after injury. Any change in the microenvironment caused by an acute injury or during a chronic disease promotes a transition from the phenotype of fibroblasts to myofibroblasts as a stimulus response (23, 24).

After a myocardial infarction (MI), the cardiac repair program in CF undergoes three distinct, but overlapping, phases: inflammatory, proliferative, and maturation (25). Resident and quiescent cardiac fibroblasts are activated 2–4 days after MI to kickstart the inflammatory phase, which is marked by the secretion of several cytokines that recruit leukocytes and, ultimately, induce the expression of proteases to degrade the ECM. During the proliferative phase, a proportion of fibroblasts differentiate into myofibroblasts and begin to proliferate and migrate from the noninjured to injured areas, increasing the production of ECM proteins. The activation of myofibroblast requires the cooperation of growth factors and proteins of the specialized matrix, which signal through the cell surface receptors to activate intracellular signaling pathways, which lead to the synthesis of proteins, such as α-smooth muscle actin (α-SMA) and the transcription of matrix macromolecules (26). The conversion of fibroblast to myofibroblast is crucial for reparative fibrosis.

When infiltrating the remodeled heart, macrophages, mast cells, and lymphocytes play an essential role in activating fibroblasts, bioactive mediators, including cytokines such as TGF-β, interleukin 10 (IL-10), and matricellular proteins (26, 27). Recent studies have shown TGF-β as an important profibrotic factor. TGF-β activation stimulates the phosphorylation of SMADs 2/3 proteins, which are transcriptional factors for collagen expression (28–30). Therefore, TGF-β is a crucial regulator of both the function and phenotype of fibroblasts, which, after stimulation and activation, are transdifferentiated into myofibroblasts (Fig. 2). This formation takes place only by the junction of associated contractile proteins such as α-SMA and nonmuscle myosin (27, 31, 32).

Figure 2.

Stimulus inducing activation of fibroblasts promoting transdifferentiation into myofibroblasts in cardiac fibrosis (CF). α-SMA, α-smooth muscle actin; MMP, matrix metalloproteinase; SMAD, mothers against decapentaplegic; TGF-β, transforming growth factor-β; TIMP, tissue inhibitors of metalloproteinase.

In addition to these functions described earlier, TGF-β modulates the activity of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), these adaptations will modify the balance between the signals of matrix preservation and degradation, favoring the fibrotic profile (31, 33).

Finally, the maturation phase is characterized by new cell transdifferentiation, this time from myofibroblast to matrifibrocytes, expressing bone and cartilage-associated ECM genes to maintain the homeostasis of the mature scar (34, 35).

MOLECULAR MECHANISMS IN CARDIAC FIBROSIS

The TGF-β pathway is one of the main pathways in CF and is associated with the transition from mesenchymal to epithelial cells (36), as well as in the process of developing tissue fibrosis, activating the canonical signal transduction pathway of TGF-β, the SMAD-2, SMAD-3, and SMAD-4 (37).

In addition to the main TGF-β/SMAD-2 signaling pathway, other molecular mechanisms are activating the cardiac fibrotic process and are interconnected to this pathway, as well as the activation of phosphoinositol-3 kinase (PI3K) and PI3K/PDK1/AKT, and the latter protein inhibits glycogen synthase kinase-3β (GSK-3β), which is a suppressor of SMAD-3; thus, SMAD-3 is phosphorylated and favors fibrotic remodeling (38).

Another signaling pathway involved in the fibrotic process is Wnt/GSK-3β/β-catenin, in which Wnt binds to the secreted Frizzled related protein 2 (sFRP2) that activates the Dsh protein family (from ‘dishevelled’). This protein inhibits GSK-3β, favoring the accumulation of β-catenin in the cytoplasm (39). This is translocated to the cell nucleus, activating the transcription of genes that induce the differentiation of cardiac fibroblasts into myofibroblasts (40). β-Catenin also phosphorylates SMAD-3, performing interconnection with the canonical pathway of TGF-β that activates SMAD, and leads to fibrogenesis and increased tissue fibrosis (41).

In contrast, the peroxisome proliferator-activated receptor-γ (PPAR-γ) inhibits TGF-β signaling (42). This mechanism is involved in this attenuation through the inhibition of α-SMA expression and by the activation of SMAD-7, which is an inhibitor of SMAD-2 and SMAD-3 (43). PPAR-γ inhibits the PI3K/AKT pathway (44) by stimulating phosphatase and tensin homolog (PTEN) phosphatase, activating GSK-3β, which in turn represses SMAD-3. Furthermore, PPAR-γ also inhibits the Wnt signaling pathway through the activation of the Dickkopf-1 protein (DKK-1), preventing the accumulation of β-catenin in the cytoplasm, enhancing its antifibrotic effect (45). On the other hand, the activation of fibroblasts promoted by the TGF-β pathway associated with the modulation of the other intracellular pathways (GSK-3β and Wnt) leads to the differentiation of these cells into myofibroblasts, increasing the production of various types of collagens, ECM, and modification of the gene expression of MMPs and TIMPs (46).

MMPs are proteins that can degrade some protein components of ECM, participating in several physiological processes such as tissue repair, healing, and mobilization of stem cells (47). The expression of these proteins is under strict control exercised by genetic and epigenetic factors, hormones, cytokines, and, mainly, endogenous inhibitory proteins, such as TIMPs (48).

TIMPs are proteins specialized in inhibiting more than 20 different types of MMPs (49). When deregulation or imbalance occurs in the expression of MMPs and TIMPs, there is a change in cellular homeostasis that induces the accumulation of collagen proteins and remodeling of the ECM leading to loss of the structural integrity of the tissue, characterizing a pathophysiological process triggering different diseases, such as cardiomyopathies (50). All these mechanisms that induce ECM remodeling and CF undergo refined control regulated by miRs.

MiRNAs IN CARDIAC FIBROSIS

Some molecular mechanisms regulated by miRNAs in the process of CF have been described, however, other mechanisms remain unknown. One of the first discovered mechanisms regulated by miRNAs in CF was through miR-21, which was upregulated in a mouse model of cardiac failure, inhibiting the expression of the sprouty homolog 1 (SPRY-1) increasing the activation of the ERK-MAP kinase pathway, inducing the fibroblasts survival and activation, fibrosis, remodeling, and cardiac dysfunction (51). In another CF study, the miR-21 also had an increased expression, however, regulating another target, the SMAD-7 gene, inhibited its expression and consequently increased the expression of the TGF-β, collagen type I (COL1A1), and COL3A1 genes, inducing atrial fibrosis and myocardial remodeling (52). Likewise, another study showed that miR-21 was also overexpressed in the heart of infarcted mice, inducing activation of cardiac fibroblasts and elevation of TGF-β expression, resulting in increased COL1A1, α-SMA, and F-actin promoting CF (53). The TGF-β signaling pathway after MI in mice also induces the inhibition of miR-29 expression with a subsequent increase in the expression of COL1A1, COL1A2, and COL3A1 collaborating to increase the collagen content in the infarcted area (54). TGF-β upregulation also induces a reduction in miR-133 and miR-590 expression, favoring increased collagen content and CF (10).

Another miRNA of the 133 family, miR-133a, is also downregulated in CF, inducing increased expression of TGF-β, connective tissue growth factor (CTGF), endothelin-1 (ET-1), and angiotensinogen (AGT) that promote increased expression of fibroblast growth factor-1 (FGF), fibronectin (FN), and COL4A1 (55). The increased expression of these genes favors the accumulation of fibrosis and myocardial remodeling.

CF was also shown in a model of MI in Sprague–Dawley rats in which reduced expression of miR-101a was identified, promoting the upregulation of TGF-β and transforming growth factor-β1 receptor 1 (TGF-βR1), inducing hypoxia, which is a common process in CF (56). MiR-22 is also downregulated in the coronary artery occlusion mice model, increasing the expression of TGF-βR1 leading to myocardial fibrosis (57).

In this context, miR-214-3p is poorly expressed in the hearts of patients with coronary artery disease and in human atrial fibroblasts stimulated with angiotensin II, generating overexpression of TGF-βR1 contributing to atrial fibrosis (58). On the other hand, miR-23b-3p and miR-27b-3p are overexpressed in atrial tissues of patients with atrial fibrillation inducing the reduction of transforming growth factor-β1 receptor 3 (TGF-βR3) with consequent elevation of COL1A1, COL3A1, and FN-1, favoring atrial fibrosis (59). Other miRNAs that have been described as associated with the CF process, are the miR-30 family, specifically miR-30b and miR-30c, all of them obtained reduced expression in this context, and these regulate the expression of the CTGF gene, which was upregulated, and has a primordial function in the structural alterations of the ECM of the myocardial tissue, converging to CF (5).

Thus, it demonstrates the importance of the miRNAs in the regulation of various genes (Table 1) and signaling pathways in the CF, requiring new strategies for the prevention and attenuation of the CF. In this context, the current evidence points to ExT as an excellent nonpharmacological strategy for the prevention and treatment of cardiovascular disease including reducing CF. Moreover, ExT also modulates the expression of several miRNAs, being an important tool to combat CF.

Table 1.

MiRNAs dysregulated in CF according to its targets

	miRNA	Target	References
↑ ↓	miR-21, miR-155 miR-133, miR-29b, miR-101, miR-221, miR-222, miR-675	TGF-β1	(10, 52, 55, 56, 60–63)
↑ ↓	miR-328, miR-23b-3p, miR-27b-3p miR-590, miR-101, miR-22, miR-98, miR-214-3p	TGF-βR1, TGF-βR2, TGF-βR3	(2, 10, 56–59, 64)
↓	miR-133, miR-30, miR-30b, miR-30c	CTGF	(5, 55, 65)
↑ ↓	miR-99b-3p, miR-199a-5p miR-26a	GSK-3β	(66–68)
↑	miR-125b, miR-503	Apelin, Apelin-13	(69, 70)
↓	miR-29	COL1A1, COL1A2, COL3A1	(54)
↑	miR-21, miR-96-5p	SMAD-7	(53, 71)
↑	miR-21	SPRY-1	(51)
↑	miR-27b-3p	FGF-1	(72)
↑	miR-323a-3p	TIMP-3	(73)
↓	miR-150	c-Myb	(74)
↑	miR-22	CAV3	(75)
↑ ↓	miR-142-3p miR-25	HMGB1	(76, 77)
↑	miR-21	ERK	(78)
↓	miR-26a	FoxO1	(68)
↑	miR-409-3p	GATA-2	(79)
↓	miR-19	MAPK	(80)
↓	miR-378	MKK6	(81)
↑	miR-125b	p53	(69)
↓	miR-24-3p	PHB2	(82)
↑	miR-130a	PPAR-γ	(83)
↑	miR-34a	PPP1R10	(84)
↓	miR-101a, miR-101b	c-FOS	(85)
↓	miR-24	Furin	(86)
↓	miR-30d	MAP4K4	(87)
↓	miR-132	MeCP2	(88)
↑	miR-494-3p	PTEN	(89)
↑	miR-223	RASA1	(90)
↓	miR-133	SRF	(65)

Effects of cardiac fibrosis (CF) on the expression of microRNAs (miRNAs): ↓upregulated miRNAs and ↓downregulated miRNAs. COL1, collagen type I; CTGF, connective tissue growth factor; FGF, fibroblast growth factor; GSK-3β, glycogen synthase kinase-3β; PPAR-γ, peroxisome proliferator-activated receptor-γ; SMAD, mothers against decapentaplegic; SPRY-1, sprouty homolog 1; TGF-β, transforming growth factor-β; TGF-βR1, transforming growth factor-β1 receptor 1; TIMP, tissue inhibitors of metalloproteinase.

MiRNAs, CARDIAC FIBROSIS, AND EXERCISE TRAINING

There are biochemical, cellular, and molecular processes that respond to different physiological stimuli determining the level of the development of different phenotypes of cardiac hypertrophy and the contribution of ExT has been extremely important in these analyses (91). Although there is controversial evidence of detrimental effects (92, 93) of endurance training and cardiac maladaptation in lifelong high-level athletes, the positive relation between ExT and CF has been seen in observational (94) and experimental studies (95). An interesting study showed that ExT used before myocardial injury (in a preconditioning design) alleviates CF and dysfunction in mice (96). ExT promotes beneficial adaptations, such as physiological cardiac hypertrophy, through regulating miRNAs (97). The physiological cardiac remodeling through ExT involving several miRNAs has been extensively studied (98–100). Interval training, a type of ExT that consists of short bouts at higher intensities (101), induced physiological cardiac hypertrophy and downregulated miR-1 and miR-133, in mice submitted to treadmill ExT (102). Intriguingly, these miRNAs were also downregulated in pathological cardiac hypertrophy. It has been also observed that the expression of miR-1 and miR-133a/b was reduced in the eccentric cardiac hypertrophy after performing two different swimming training protocols when compared with sedentary controls (103). In addition, another study revealed that the expression of CTGF is controlled by miR-30 and miR-133 in human and rodent cardiac tissue (5). Thus, the miRNAs involved in one of the major fibrosis-promoting factors, the CTGF, are regulated by ExT.

A recent study observed that when miR-122 is downregulated in the heart, there is a possibility that TGF-β1 is upregulated being involved in myocardial fibrosis in aortic stenosis (104). Although ExT has not been linked to this miRNA, other analyses showed that ExT could target genes that are involved in programmed TGF-β signaling (105). TGF-β mediates the reduced expression of the miR-29a, -29b, and -29c and their target genes related to CF (97).

It has been reported that swimming training is capable of rescuing miR-29a, and miR-29c expression in the cardiac muscle of myocardial-infarcted rats (106). ExT increases miR-29c and favors the reduction of the collagen concentration in the heart and improves the functioning of the left ventricle inducing an antifibrosis effect (54). Upregulation of miR-29b and miR-455 was also observed in the heart of exercised diabetic mice suggesting that ExT for 8 wk, 5 days/wk on a treadmill can mitigate the deleterious effects of this pathological condition (107). These results together indicate that ExT regulates miR-29 families contributing to reducing the levels of TGF-β.

Downregulated expressions of miR-208a and miR-208b were seen in the ExT group when compared with the sedentary group, promoting the reduction of myosin heavy chain (MHC) expression, and inducing increased cardiac compliance (98, 108). Another study suggests that the reduced expression of miR-208 could mediate the positive effects of ExT against cardiovascular disease (CVD) (109). It has also been reported a reduction in the expression of miR-26a and an increase in miR-150 after voluntary wheel running exercise (110). These genes are involved in the cardiac physiological adaptations that lead to hypertrophy induced by ExT (Table 2).

Table 2.

MiRNAs altered expressions in heart after ExT according to its targets

	miRNA	Target	References
↑ ↓	miR-29a, miR-29c, miR-101a miR-1, miR-133a, miR133b	COL1A1, COL1A2, COL3A1	(103, 111–114)
↑	miR-29a	TGF-β1	(111)
↑	miR-150	GSK3β	(110)
↑	miR-17-3p	TIMP-3	(115)
↑	miR-126	HIF-1α	(116)
↓	miR-99b, miR-100	IGF-1	(117)
↓	miR-26b	IGF-1R	(110)
↑	miR-21	PDCD4	(118)
↓	miR-124	PIK3α	(119)
↑	miR-126	VEGF	(113, 120)
↑	miR-499, miR-206	VEGFα	(121)
↑	miR-145	Wnt3a	(122)
↑	miR-486, miR-17-3p, miR-21, miR-144, miR-19b	PTEN	(115, 119, 123)
↑	miR-101a	Fos	(111)
↑	miR-126	SPRED2	(124, 125)
↑	miR-27a	ACE	(126, 127)
↓	miR-143	ACE2	(127)
↓	miR-27, miR-208	GATA-4	(110, 121)
↑	miR-19b, miR-30	BCL-2	(117, 118)
↓	miR-1
↑	miR-126	PI3K, PI3KR2	(120, 124, 125, 128)
↑	miR-155	AT1R	(126)
↑	miR-145	Dab-2	(122)
↑	miR-486	FOXO	(123)
↓	miR-27, miR-208	GJA1	(121)
↑	miR-222	HIPK1	(129)
↑	miR-1	NCX	(130)
↓	miR-214
↑	miR-30b	p53	(117, 118)
↓	miR-191a
↑	miR-126	Raf	(120)
↑	miR-382-3p	Resistin	(131)
↑	miR-1	SERCA-2a	(130)
↓	miR-214
↑	miR-145	TSC2	(119)

↑Upregulated microRNAs (miRNAs). ↓Downregulated miRNAs. COL1, collagen type I; GSK-3β, glycogen synthase kinase-3β; HIF, hypoxia-inducible factor; PPAR-γ, peroxisome proliferator-activated receptor-γ; TGF-β, transforming growth factor-β; TGF-βR1, transforming growth factor-β1 receptor 1; TIMP, tissue inhibitors of metalloproteinase.

Another cardiac physiological adaptation was observed in the miR-21 and miR-144 expression related to a reduction in their target gene, and a greater expression of miR-145 followed by a reduction in tuberous sclerosis complex was also identified (119). Conversely, the authors also showed that a reduction in cardiac miR-124 expression by ExT is associated with an increase in their target gene (PI3K) inducing physiological hypertrophy.

MiR-222 and miR-17-3p are necessary for the induction of cardiac growth in the exercised heart (115, 129), but there is no research involving the regulation of this miRNA. In addition, it has been reported that miR-503 contributes to an increased expression of angiotensin II leading to CF (70) and ExT effects were not investigated either. It is known that ExT in cardiac hypertrophy in normotensive rats can increase miR-27a and miR-27b expressions targeting angiotensin-converting enzyme (127). Conversely, in this same study, it was observed a decrease in miR-143 expression.

Finally, swimming training could induce cardiac hypertrophy, indicating that miR-99b and miR-100 were reduced, whereas miR-30e, miR-133b, and miR-208 were increased (117). Other miRNAs were also shown to be dysregulated by ExT as seen in Table 2. ExT appears as a possible way to combat the main gene targets and signaling pathways regulated by miRNAs responsible for the CF. These new findings could unveil targets to develop miRNA-based therapies.

OVERLAP MiRNAs IN CARDIAC FIBROSIS AND EXERCISE TRAINING

Furthermore, we performed an analysis of the miRNAs expressed in both contexts: CF and ExT, creating a Venn diagram. In this Venn diagram, we see that 42 miRNAs were dysregulated in CF and 83 miRNAs were dysregulated in the ExT scenario. Interestingly, we identified that 22 miRNAs are overlapping in both conditions, they are miR-21, miR-22, miR-23b, miR-24, miR-27b, miR-29a, miR-29b, miR-29c, miR-30b, miR-34a, miR-99b, miR-101a, miR-125b, miR-130a, miR-132, miR-133a, miR-155, miR-199a, miR-214, miR-221, miR-222, and miR-223.

Even more important, we observed that of these 22 miRNAs that are overlapping, eight miRNAs have opposite expression patterns in CF and ExT, they are: miR-24, miR-29b, miR-29c, miR-30b, miR-101a, miR-132, miR-221, and miR-222. Coincidentally, all miRNAs in the CF process are downregulated and all miRNAs in ExT are upregulated (Fig. 3).

Figure 3.

Overlapping between microRNAs (miRNAs) in cardiac fibrosis (CF) and exercise training (ExT). Red line box contains all the 22 overlapping miRNAs found in both CF and ExT. MiRNAs with divergent expressions in these conditions are expressed in bold letter. ↓Downregulated miRNAs in CF. ↑Upregulated miRNAs in ExT.

The miRs-29b, -29c, -101a, -221, and -222 are regulators of TGF-β expression. In CF they have reduced expression promoting TGF-β overexpression with increased collagen expression. miR-30b is also attenuated in CF, inducing an increase in CTGF, which is another gene that contributes to CF and cardiac remodeling. Likewise, the reduced expression of miR-101a promotes the increase of the c-FOS gene, which favors cell proliferation and differentiation, collaborating with the CF process. In this context, downregulated miR-132 also promotes increased expression of its target genes Ras-GTPase activating protein and methyl-CpG-binding protein 2, inducing attenuation of angiogenesis, decreased survival, and increased fibrotic activity.

Thus, as it was observed that these same miRNAs are overexpressed in an animal model after ExT, we can suggest that ExT possibly modulates the expression of these miRNAs beneficially in the context of CF, attenuating the signaling pathways of these specific miRNAs, inducing improvement in the phenotype and cardiac function with probable CF attenuation, however, studies are needed to prove this hypothesis.

BIOINFORMATICS PREDICTION OF SELECTED MiRNAs

To complement the understanding of these eight miRNAs that have different expression patterns in CF and ExT in the Venn diagram, including the different target genes and signaling pathways that they are regulating, we performed a bioinformatics analysis using the miRWalk tool that stores predicted data obtained with a machine learning algorithm that includes miR-target interactions (132). For each miRNA, a graph was generated in the miRWalk tool integrated miRbase and informatics.jax.org with the various target genes that are being regulated (133), however, we only applied it to validated targets, as can be seen in Fig. 4.

Figure 4.

Bioinformatic analysis of eight microRNAs (miRNAs) and all its validated target genes by miRWalk tool.

Subsequently, to identify the signaling pathways as well as the biological processes regulated by these eight miRNAs, we performed an exploratory analysis of these eight miRNAs in both 5p and 3p, by DIANA Tools miRPath v.3 (134) integrated with TargetScan with GO analysis (135) (Fig. 5) and TarBase with GO analysis (136; Fig. 6) to identify predict and validated targets, respectively, and involved in associated biological process.

Figure 5.

Bioinformatic analysis of signaling pathways and biological processes regulated by the eight microRNAs (miRNAs) using TargetScan with GO analysis.

Figure 6.

Bioinformatic analysis of signaling pathways and biological processes regulated by the eight microRNAs (miRNAs) using TarBase with GO analysis.

In this first heatmap analysis we can see that miR-29b-3p and miR-29c-3p are regulating several biological processes, such as extracellular matrix, proteinaceous and structural constituents of the extracellular matrix, fiber organization, and collagen trimer, COL4A1, in addition to anatomical structural development, demonstrating the importance of these miRNAs during the fibrotic process and their downregulation with a consequent increase in collagen expression are important factors contributing to cardiac remodeling and mainly to decrease cardiac function. The dysregulation of miR-24-3p, -24-1-5p, -101a-3p, -221-5p, and -222-5p is also demonstrated in this analysis that promotes changes in anatomical structural development in addition to altering intracellular signaling pathways. On the other hand, ExT can increase the expression of these miRNAs by attenuating the expression of collagen genes with a consequent reduction in cardiac collagen content, promoting improved heart function.

In this second heatmap analysis, we observed that dysregulation of the miR-24-3p, -29 b-3p, -29c-3p, -30 b-5p, -101a-3p, -132-3p, -221-3p, and 222-3p are associated with the modification of the pattern of anatomical structural development, in addition to being associated with the process of cellular protein modification, alteration in the chromosomal and cytoplasmic organization, changes in biological processes and with changes in cell morphology and intracellular signaling pathways in both CF and ExT, which is why we can see these categories very altered in the heatmap. These bioinformatics results reinforce our explanation in this review, showing that CF promotes changes in the expression of these miRNAs with consequent alteration in the cardiac phenotype generating pathological remodeling of the heart and that ExT can change the expression pattern of these miRNAs inducing a beneficial change in the cardiac phenotype with improved function.

CONCLUSIONS

CF plays an important role in heart remodeling and dysfunction after a cardiac injury, while ExT is a potent nonpharmacological agent in overall cardioprotection. Despite the differences in pathophysiological processes between rodents and humans, this study provides an insight, from animal studies, that miRNAs associated with both CF and ExT, mainly those with inverse expressions, and its up- and downstream pathways could explain how ExT attenuates the fibrogenic process at molecular level.

This study presents, based on the literature associated with bioinformatic analysis, miRNAs involved in the CF process and related to ExT, highlighting miR-24-3p, -29 b-3p, -29c-3p, -30 b-5p, -101a-3p, -132-3p, -221-3p, and 222-3p, opening a path of investigation for future experimental and clinical studies as potential biomarkers and eventually unveil therapeutic targets to treat cardiac disorders.

Finally, these miRNAs that are oppositely modulated by ExT could be tested as gene therapy to attenuate CF in individuals who cannot exercise, mimicking the effect of ExT without needing to exercise.

GRANTS

The researchers were supported by Sao Paulo Research Foundation Grants 2015/22814-5, 2018/22579-4, 2021/06229-6, 2022/00531-5, 2022/02339-4, and 2022/03138-2; National Council for Scientific and Technological Development Grants 313376/2021-2, 200723/2020-0, 422514/2021-7, and 409629/2021-9; and Coordination for the Improvement of Higher Education Personnel Grants 88887.484856/2020-00 and 88887.804476/2023-00.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.C.I.-C., L.F.R., V.H.A.J., R.A.L.D.S., T.F., and E.M.O. analyzed data; A.C.I.-C., L.F.R., and V.H.A.J. prepared figures; A.C.I.-C., L.F.R., V.H.A.J., and R.A.L.D.S. drafted manuscript; A.C.I.-C., L.F.R., V.H.A.J., R.A.L.D.S., T.F., and E.M.O. edited and revised manuscript; A.C.I.-C., L.F.R., V.H.A.J., R.A.L.D.S., T.F., and E.M.O. approved final version of manuscript.