Heart muscle can continue to die even after restoring blood following a heart attack but a tiny RNA can helps the heart

Heart muscle can continue to die even after restoring blood following a heart attack, and scientists have new evidence that one way to help it live is by boosting levels of a tiny RNA that helped the heart form.

In their mouse model of this ischemia/reperfusion injury of the heart, they’ve found they can reduce heart muscle death 40 percent by giving a manmade version of the microRNA miR322, they report in the Journal of Molecular and Cellular Cardiology.

MiR322 is known to coax stem cells to make heart cells during development but is typically found at a lower, more basal level and with an unclear purpose in the adult heart, says Dr. Yaoliang Tang, cardiovascular researcher in the Vascular Biology Center and Department of Medicine at the Medical College of Georgia at Augusta University.

Tang and his team were looking for better ways to protect the heart from additional damage, doing high throughput analysis of microRNAs, which help regulate gene function, and miR322 was the first standout as the most dramatically reduced after blood flow to the heart was restored.

So Tang’s team began looking for the source of miR322 in adult hearts – which turned out to be the endothelial cells that line the blood vessels – and in the process they also found miR322’s target.

For these studies, they caused an occlusion in the left coronary artery of mice for about 45 minutes, then enabled reperfusion to reflect what happens when a human has a heart attack, then gets treatment.

During the period of ischemia – when the heart attack happens – they saw seriously reduced miR322 levels, which recovered after blood and oxygen were restored but dropped dramatically again one day later, the lowest point was seven days later.

When they increased miR322 by giving what scientists call its mimic, they saw levels of the endogenous heart cell protector Notch 1 significantly increase while levels of cell suicide promoter FBXW7 significantly decreased, and heart damage reduced.

When they gave both miR322 mimic and FBXW7 plasmid – a plasmid is small DNA molecule, which can be used to increase expression of a gene – Notch 1 levels instead decreased and the heart benefit was lost, implicating FBXW7 as a direct target of miR322.

“We still need to see how and if it can protect the heart long term, but we find in the short term when we give enough of this microRNA, it protects the heart from reperfusion injury,” Tang says.

While the half-life of miR322 is just a few seconds and it’s very expensive, it likely could one day be used in the immediate aftermath of a heart attack to reduce permanent heart damage, Tang says.

A great therapeutic advantage is that it’s so small it’s easily taken up by heart cells, he adds.

Still a better option might be finding a way to instead bolster the body’s natural method for increasing miR322, which the team is now looking to find.

Heart disease is the leading cause of death in Georgia and the United States, according to the Centers for Disease Control and Prevention.

Inadequate blood and oxygen to the heart, or ischemia, resulting from a clot or other occlusion of a big blood vessel in the heart, is the primary problem.

However solutions, including cardiac bypass surgery and angioplasty to reopen blocked coronary arteries, can result in a second period of adjustment and injury that can be responsible for as much as half of the size of the damage done to the heart muscle, called the infarct size.

If it seems odd that reestablishing blood rich in oxygen and nutrients back to heart cells that are screaming for both and dying without them, would also cause injury, Tang explains that it’s a fragile transition period.

When oxygen levels drop, heart cells’ metabolic rate drops as well to reduce their needs, much like cooling the body during cardiac surgery.

“Heart cells are fragile and when oxygen reenters they need to switch back to their normal metabolic rate,” Tang says. “A lot of cells just don’t adjust well and will die.”

The state of ischemia also produces and recruits a variety of factors and cells that promote inflammation to help protect from infection and haul off debris from dying cells.

This highly inflammatory state produces a lot of free radicals, unstable atoms that can cause even more damage to heart cells.

“That is why a lot of patients don’t die because of ischemia, they die because of reperfusion,” Tang says.

Notch 1 signaling is an endogenous method of protecting the heart from oxidative stress that can prevent cell suicide – which is in direct conflict with the tagging for death work of FBXW7 – so here FBXW7 modifies active Notch 1 so it can’t function.

FBXW7 is called an ubiquitin enzyme, because it adds the small molecule ubiquitin to proteins, which basically tags them for delivery to the garbage.

“It’s a bad enzyme,” says Tang.

In this scenario, one of the things it’s modifying is active Notch 1 and when FBXW7 goes us up, miR322 goes down.

In fact, there is not just an association of the two but miR322 typically regulates FBXW7, physically binding to it to keep it from doing anything bad. When ischemia reduces miR322 levels, FBXW7 is freer to tag and eliminate, Tang says.

MiR322 overexpression, which increases Notch 1, has been shown to have similar protective effects in the brain, where an ischemic stroke has pretty much the same effect as a heart attack.

MiR322, called miR-424 in humans, was known to show up following ischemia but where it came from and what exactly it was doing remained unknown.

Next steps include studies in a larger animal model, and Tang also wants to learn more about two other microRNAs that seem to function as partners to miR322.

Because while miR322 was the first microRNA they found dramatically impacted, they would also find two others whose levels seemed to rise and fall in tandem with miR322’s. There is clearly crosstalk between them, Tang says.

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The causes of heart failure (HF) include ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), hypertension, valvular heart disease, diabetic cardiomyopathy and congenital heart disease (CHD) (1).

The pathogenesis of HF is associated with myocardial hypertrophy, fibrosis or necrosis, cardiomyocyte apoptosis, renin-angiotensin-aldosterone system imbalance and collagen changes, as well as several other factors (2–7).

MicroRNAs (miRs) are small (~22 nucleotides in length), single-strand, non-coding RNA sequences derived from precursors that control gene expression in a variety of physiological and developmental processes, which are involved in post-transcriptional regulation of gene expression (8). miR disorders are associated with a number of human diseases, including diabetes, myocardial infarction and cardiovascular disease, obesity and cancer.

Several studies have demonstrated that miRs may affect different aspects of the occurrence and development of HF (9–14).

The association between miRs and HF is discussed in detail below.

Circulating miRs are increasingly recognized as promising biomarkers, given their stability and resistance to endogenous RNase (15); these miRs, to some degree, may also be used as diagnostic biomarkers for angiocardiopathy. In addition, miRNAs and various types of HF have complex relationships, as described below.

Changes and associated mechanisms of miRs in various types of HF

miRs may be involved in several aspects of the occurrence and development of HF, such as cardiomyocyte apoptosis, hypertrophy, fibrosis, inflammation, oxidative damage and hypoxic damage (9–14), among others.

The specific regulatory functions of miRs are indicated in Figs. 1and ​and22 and are summarized in Table I (16–66).

Table I – Open in a separate window

Circulating miRs as diagnostic biomarkers

HF is primarily caused by cardiomyopathy, hypertension, diabetes and CHD, among other causes (15). The different etiology is associated with several miRs.

miRs associated with cardiomyopathy

The cardiomyopathies leading to HF predominantly include DCM and ICM (67–71). DCM, characterized by left ventricular dilatation, ventricular wall thinning and diffuse myocardial dysfunction, leads to congestive HF (72) and right ventricular dysfunction (73). These pathological changes result in the transition from compensatory hypertrophy to DCM (74). The heart undergoes continuous remodeling of myocardial cells through transduction of intercellular signals and activation of the transcription and transmission pathways (75). Naga Prasad et al (76) performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis on a set of samples used for miR microarray analysis, and identified that hsa-mir-378 (P<0.0055), hsa-mir-001 (P<0.0001), hsa-mir-007 (P<0.0009) and hsa-mir-29b (P<0.0087) were notably decreased in DCM compared with control samples; by contrast, hsa-mir-342 (P<0.0004), hsa-mir-214 (P<0.0001), hsa-mir-125b (P<0.0785), hsa-mir-145 (P<0.0091) and hsa-mir-181b (P<0.0047) were significantly increased in DCM compared with non-failing controls, and may be used to indicate the stage of HF development. Enes Coşkun et al (77) investigated 23 pediatric patients (aged 2–192 months) with isolated idiopathic DCM as the experimental group, and 26 age-matched healthy children with innocent murmur as the control group. Patients with fractional shortening of <25% and with a left ventricular end-diastolic diameter >112% of the predicted dimension were considered to have DCM. The results of RT-qPCR demonstrated that the expression levels of miR-454 and miR-518f were significantly higher in DCM patients compared with those in the control group. Furthermore, the expression levels of 10 miRs (miR-618, miR-875-3p, miR-205, miR-194, miR-302a, miR-147, miR-544, has-miR-99b, miR-155 and miR-218) were notably lower in patients with DCM compared with control subjects, suggesting that they may be used as potential diagnostic biomarkers. Interestingly, Miyamoto et al (78) observed that 2 miRs (hsa-miR-636 and hsa-miR-155) were upregulated and 2 miRNAs (hsa-miR-646 and hsa-miR-639) were downregulated in patients with DCM compared with patients with DCM with recovered ventricular function, which indicated that they may serve as diagnostic as well as prognostic biomarkers. However, further research is required to elucidate the specific underlying mechanisms.

Leger et al (79) and Zeng et al (80) measured left ventricular ejection fraction (LVEF) and the 6-min walk test distance (6MWTD) and CBP/p300 interacting transactivators with ED-rich termini 2 (CITED2), hypoxia-inducible factor-1 (HIF-1) in patients with ICM before and after treatment, and identified that LVEF, 6MWTD, CITED2 and HIF-1 levels were significantly lower in the ICM group compared with those in the control group prior to treatment (P<0.01). The N-terminal pro-B-type natriuretic peptide (NT-proBNP), HIF-1 and miR-182 levels in the ICM group were significantly higher compared with those in the control group (P<0.01). Following 4 months of treatment, the levels of 6MWTD, CITED2 and LVEF in the ICM group were significantly increased, whereas the levels of plasma NT-proBNP, HIF-1 and miR-182 were significantly decreased (P<0.01). Furthermore, the plasma miR-182 level was negatively correlated with CITED2, LVEF and 6MWTD (P<0.05) and positively correlated with HIF-1 (P<0.05) in the ICM group. Therefore, miR-182 is correlated with several indicators of HF, and may be considered to reflect the severity of the disease. Olson and Rooij (81) and Fichtlscherer et al (82) observed upregulation of miR-208a and miR-499 and downregulation of the circulating levels of miR-126, miR-17, miR-92a and the inflammation-associated miR-155 in patients with coronary artery disease compared with healthy controls by qPCR. Similarly, the level of miR-145 in smooth muscle was significantly reduced. By contrast, the levels of cardiac muscle-enriched miRs (miR-133a and miR-208a) tended to be higher in patients with coronary artery disease. Li et al (83) demonstrated a decrease of miR-125a, miR-20a and miR-302d levels in ICM using Deep RNA sequencing. Notably, only 55 miRs were indicated to be consistently increased in ICM and non-ischemic cardiomyopathy (NICM), including miR-21-5p, miR-125b-1-3p and miR-106b-5p, among others. However, 38 miRNAs were downregulated in both ICM and NICM (non-ischemic cardiomyopathy), including miR-20a-5p, miR-17-5p and let-7e-5 (83). The findings suggest that miR-182 appears to be a promising new biomarker for the diagnosis of ICM and DCM in clinical research.

miRs associated with hypertension

Hypertension is an independent risk factor for cardiac and cerebrovascular disease (84). It has been reported that at least 50% of patients with long-term hypertension will likely undergo cardiac remodeling, particularly left ventricular remodeling (85). Myocardial cell hypertrophy is among the primary causes underlying the occurrence of HF (86). Notably, it has been demonstrated that miR-208 can induce cardiac hypertrophy and results in the overexpression of β-myosin heavy chain in myocardial fibrosis (87). Several miRs were indicated to be differentially expressed in hypertension, including miR-296-5p, let-7e and human cytomegalovirus (HCMV)-miR-UL112, as encoded by HCMV in previous studies of the hypertension-associated miR spectrum (88–90). Interferon regulatory factor 1, which is involved in the regulation of blood pressure by acting on nitric oxide synthase and vascular angiotensin (Ang) receptor, was demonstrated to be a direct target of HCMV-miR-UL112 (91). However, in hypertension, HCMV titers are considered to reflect the expression level of HCMV-miR-UL 112 (91), which is an independent risk factor for hypertension. HCMV has been reported to inhibit vasodilation by impairing nitric oxide synthase function (92) and causing endothelial cell dysfunction (93). However, further research is warranted due to the elusive association between HCMV infection and endothelial dysfunction.

Kontaraki et al (94,95) reported that upregulated miRs included miR-1 and miR-21, whereas downregulated miRs included miR-9, miR-126, miR-133, miR-143 and miR-145 in the hypertension group compared with the healthy control group. In addition, miR-21, miR-143 and miR-145 were negatively correlated and miR-133 was positively correlated with 24-h ambulatory mean blood pressure, mean diastolic blood pressure and mean pulse pressure in the hypertension group. Furthermore, miR-9 and miR-126 were positively correlated with mean pulse pressure, but the association between miR-9 and left ventricular hypertrophy index was positively correlated with the 24-h ambulatory mean blood pressure and mean diastolic blood pressure. Therefore, this miR may reflect the severity of hypertensive HF.

Dickinson et al (96) reported that the circulating levels of miR-423-5p, miR-106b, miR-20b, miR-223, miR-16 and miR-93 were markedly increased in hypertension-induced HF, which was confirmed via RT-qPCR analysis of plasma RNA from hypertensive rats. These results indicate that several miRs can reflect disease progression to a certain extent, and may be used as biomarkers of hypertensive HF. This suggests that miRs should be detected pre- and post-treatment to reduce the effects of medication on the results of the experiment. Hou et al (97) randomly divided 16 spontaneously hypertensive rats (SHR) into the SHR control (distilled water) and intervention SHR (captopril 10 mg/kg/day) groups. An additional 8 Wistar male rats comprised the normal control groups (captopril 10 mg/kg/day or distilled water for 8 weeks). The expression of miR-137 was detected by RT-qPCR and western blot analysis in rat hearts, and miR-137, Ang II, transforming growth factor (TGF)-β1, Smad3, collagen (Col)-I and Col-III were identified to be more highly expressed in the SHR treatment and SHR control groups than the normal control group (P<0.01 and P<0.05, respectively); by contrast, the levels of miR-137, Ang II, TGF-β1, Smad3, Col-I and Col-III were significantly lower in the normal control groups compared with the SHR control group (P<0.01 and P<0.05, respectively). Thus, miR-137 may promote cardiac remodeling in SHR by upregulation of Ang II and the TGF-β1/Smad3 signaling pathway; in addition, captopril intervention can inhibit miR-137 expression. Therefore, miR-137 not only indicates the presence of high blood pressure, it may also reflect its severity.

Li et al (98) reported that insulin-like growth factor (IGF)-1 prevented diabetes-induced cardiomyopathy via marked anti-apoptotic and anti-fibrotic effects, which are mediated by miR-1. These findings provide a new paradigm for the endocrine effects of IGF-1 in the heart, and suggest that cardiac-specific miR-1 may be a useful biomarker and therapeutic target for diabetes-induced cardiomyopathy. Yang et al (99) observed that miR-505 interfered with the migration of cultured endothelial cells through targeting fibroblast growth factor 18, suggesting that miR-505 may be involved in vascular regeneration. In addition, a group of miRs (miR-92a, miR-130a and miR-195) were demonstrated to be abnormally expressed in hypertensive patients with metabolic syndrome. Notably, miR-92a is differentially expressed in the blood of hypertensive and non-hypertensive patients (100) and may promote miR-mediated intercellular communication (101). Kontaraki et al (94,95) confirmed several types of differentially expressed miRs in an animal model: Myocardial hypertrophy was induced by miR-21, miR-208b and miR-499; the anti-myocardial hypertrophy miRs comprised miR-1, miR-26b and miR-133a, of which miR-1, miR-21, miR-208b and miR-499 were upregulated, whereas miR-26b and miR-133a were downregulated in peripheral blood mononuclear cells from patients with hypertension compared with healthy controls. In patients with hypertension, the degree of left ventricular hypertrophy was negatively correlated with the miR-1 and miR-133 indices, whereas the miR-21, miR-26b, miR-208b and miR-499 indices were positively associated with left ventricular hypertrophy.

miRs associated with diabetic HF

Dickstein (102) reported that the occurrence and development of insulin resistance in HF was correlated with overactivation of the renin-angiotensin-aldosterone system (103,104), disturbance of energy metabolism in the myocardium (105), liver pathology, as well as other factors. It was previously demonstrated that the expression of miR-133 and miR-1 increased significantly in myocardial cells following hyperglycemic injury (106). IGF-1 and IGF-1 receptor are the two target genes of miR-1 (107). Previous studies demonstrated an increasing level of miR-133 and decreasing levels of miR-650, miR-222 and miR-338 in hyperglycemic cardiomyocyte injury (108,109). Greco et al (110) collected biopsies from the peri-infarctual area (border zone) and the non-ischemic remote zone from patients with diabetic HF (D-HF), non-diabetic HF (ND-HF) and the control group. miR expression was measured using RT-qPCR in left ventricular biopsies from 10 patients with D-HF and 19 patients with ND-HF affected by non-end-stage ischemic cardiomyopathy. A total of 17 miRs were revealed to be differentially expressed in patients with D-HF and/or ND-HF when compared with control subjects; in particular, miR-34b, miR-34c, miR-210, miR-199b and miR-372 were upregulated, whereas miR-650 and miR-223 were downregulated. Therefore, miRs may not only be obtained from the blood or serum, but also from tissue biopsies, when the content in the body fluids is low. Nandi et al (111) and Deng et al (112) reported that attenuation of miR-133a in diabetic hearts is associated with the induction of autophagy and hypertrophy. In conclusion, attenuation of miR-133a appears to serve a key role in D-HF and contributes to the exacerbation of diabetes-mediated cardiac autophagy and hypertrophy in patients with HF undergoing left ventricular assist device implantation. Chavali et al (113) used multiplex RT-qPCR in insulin 2 mutant Akita mouse hearts (a diabetic mouse model with heart disease) and observed marked downregulation of miR-744, miR-142-3p, miR-384-3p, miR-494, let-7a, miR-450, miR-338, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-433, miR-451, miR-455, miR-500, miR-542-3p and miR-872. By contrast, miR-295 was upregulated in Akita mouse hearts. Therefore, miR-295 may be used as a mammalian-specific miR in early embryonic stages. Increased miR-295 expression was associated with pathological changes in Akita mouse hearts. miR-223, as an anti-inflammatory miR, may reflect the progression of diabetic Ins2+/− Akita heart disease or D-HF. In another study, miR-1 and miR-133A were demonstrated to act as regulators of glucose homeostasis in vitro (113). Notably, miR-133a/b can reduce the expression of glucose transporter 4 and inhibit the uptake of glucose by insulin-induced myocardial cells (114). Furthermore, miR-133a/b targets Kruppel-like transcription factor 15, which is directly involved in this process (108). Two other target genes of miR-133a/b are the human ether-a-go-go-related gene and KCNQI, and these two genes are involved in the regulation of cardiac K+ channels and the presence of long QT syndrome in patients with diabetes (115). The decrease of miR-126 in diabetic microvascular tissues may indicate the severity of diabetic vascular complications (116); however, the expression of miR-126 did not decrease, but was rather significantly increased in patients with coronary atherosclerosis (117). In a mouse model of type 1 diabetes mellitus established by streptozotocin (118), 15 miRs were differentially expressed in the myocardium, among which 10 miRs (miR-195, miR-199a-3P, miR-700, miR-142-3p, miR-24, miR-21, miR-22, miR-499-3p, miR-208a and miR-705) were upregulated, whereas 5 miRs (miR-29a, miR-1, miR-373, miR-143 and miR-20a) were notably downregulated. Histological examination revealed hypertrophy of the myocardial cells in type 1 diabetes mellitus group mice compared with the control group, with a disorderly arrangement and enlarged nuclei. Notably, the prediction of associated target genes primarily involves cell growth, differentiation, proliferation, collagen fiber growth, apoptosis and angiogenesis.

miRs of HF in CHD

CHD is a multi-gene genetic disease resulting from structural or functional cardiovascular abnormalities present at birth that are caused by congenital abnormalities (119). Disrupted miR expression may result in CHD via specific protein regulation. miR-133 and miR-1 are present in the same transcription unit (120); miR-1 is the most abundant miR and is highly conserved in human myocardial cells (121). Mature miR-1-1 and miR-1-2 have the same gene sequence; the miR-13 family includes miR-133a-1, miR-133a-2 and miR-133b (122,123). During heart development, the deletion or mutation of the essential gene Hand2 of muscle precursor cells in early embryonic development may lead to cardiac hypoplasia and even cardiac arrest (124). Mukai et al (125) revealed that miR-486-3p, miR-155-5p and miR-486-5p were increased in patients with cyanotic heart disease compared with those without heart disease. Furthermore, let-7e-5p and miR-1260a were decreased in patients with early-stage acyanotic heart disease compared with those without heart disease, suggesting that these miRs may be used for early diagnosis.

Zhao et al (126) reported that the expression of miR-1-2 was upregulated in myocardial and skeletal muscle cells. Overexpression of miR-1 during cardiac development may inhibit ventricular myocyte dilatation. It was also demonstrated that miR-1-2 targets the Hand2 gene, which may block Hand2 protein synthesis and regulate cardiac morphogenesis (127); its abnormal expression may even lead to CHD (127). Another study reported that the mouse phenotypes were almost normal with deletion of either miR-133a-1 or miR-133a-2, but the synchronous lack of these two miRs led to a fatal ventricular septal defect in approximately half of the mice during the embryonic period (128). Thus, miR-133 can promote myoblast proliferation, and miR-1 can stimulate myogenic differentiation. Therefore, miR-1 and miR-133 exhibit a dialectical association, and abnormalities may lead to the development of CHD.

Chen and Li (129) quantified the levels of miR-19a by RT-qPCR in the plasma of 30 patients with CHD, and changes in the levels of miR-19a, miR-130a and miR-27b were also confirmed using RT-qPCR. The levels of miR-19a, miR-198, miR-130a and miR-27b were significantly increased in patients suffering from pulmonary arterial hypertension induced by CHD. These observations suggest that circulating miR-19a may be a novel biomarker for the diagnosis of pulmonary arterial hypertension induced by CHD.

The abovementioned data summarize the differences in expression of miRs in patients with HF (including cardiomyopathy, hypertension, D-HF and CHD). Their clinical significance as HF biomarkers were analyzed (Table II).

Limitations of miRs as biomarkers of HF

Establishing an accurate, reliable circulating miR system for HF diagnosis, prognosis and prediction of response to treatment is challenging, from sample collection and processing to data analysis. First, overlapping between various failure mechanisms leads to difficulties in assessing which mechanisms underlie the expression changes in circulating miRs. Second, serum or plasma are the first choices for sample selection and handling, but the level of circulating biomarker miRs was low, which to some degree impedes the detection of miRs (130). Serum hemolysis may result in waste of samples (131). Furthermore, the serum level of miRs was higher than for circulating plasma, indicating that serum samples can prevent potential interference caused by platelets and leukocytes during sample preparation (132). Therefore, use of the same type of material and synchronous sampling is important for the patient and control groups, as well as a standard scheme to avoid sample hemolysis, minimizing differences between patient selection and classification. Third, some studies have reported fluctuation of miR levels in patients with HF following treatment (133,134). Blood samples were collected at three stages, namely prior to, during and following treatment. A fourth factor was the choice of measurement platform for miR. As indicated in Fig. 2, all research techniques have advantages and disadvantages, but the most commonly used method is RT-qPCR. This method is more sensitive and more cost-effective compared with other methods, but its primary limitation is the inability to detect new miRs. In addition, the standardization of miR expression level may be difficult, as the expression levels of miRs fluctuate with changes in physiological and pathological conditions. Therefore, standard methods are commonly used for the experiments, including the use of equal amounts of starting material (such as serum or plasma), which is more reliable for endogenous miRs for data normalization.

As observed in the present study, the clinical manifestations of HF caused by expression changes of different miRs are similar, and the changes in miR expression caused by different types of HF may also be similar, reflecting the complexity of miR biology.

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More information: Zixin Chen et al, MiR322 mediates cardioprotection against ischemia/reperfusion injury via FBXW7/notch pathway, Journal of Molecular and Cellular Cardiology (2019). DOI: 10.1016/j.yjmcc.2019.05.020

Journal information: Journal of Molecular and Cellular Cardiology
Provided by Medical College of Georgia at Augusta University