Disease severity–related alterations of cardiac microRNAs in experimental pulmonary hypertension

Abstract Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart‐relevant microRNAs, that is myomiRs (miR‐1, miR‐133a, miR‐208, miR‐499) and miR‐214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT‐qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR‐1/miR‐133a/miR‐208a/miR‐499) were decreased and the expression of miR‐214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR‐214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.

induces a reliable model of experimental pulmonary hypertension (PH) which mimics a number of features of human PAH. [5][6][7][8] MicroRNAs (miRNAs) are small non-coding RNA molecules (around 22 nucleotides long) that regulate gene expression on post-transcriptional level, by mRNA degradation or translational repression 9 and various miRNAs have been suggested to be involved in the pathophysiology of PAH. 10,11 Importantly, alterations in muscle-specific microRNAs (myomiRs), that is miR-1, miR-133a, miR-208 and miR-499, have been found in diverse cardiac injuries 12,13 and, recently, myomiRs have been unveiled to be sensitive markers of sudden death from myocardial infarction. 14 Thus, their role in RV pathology resulting from PAH can be anticipated.
MiR-1 is abundantly expressed in the skeletal muscle and the heart and is a key regulator in differentiation and proliferation of muscular cells. 13 MiR-1 also plays a crucial role during cardiogenesis and regulates cardiac conduction. 15 Its increased levels have been detected in patients with coronary arterial disease and in rat hearts after myocardial infarction. 16 On the contrary, decreased levels were observed in patients with atrial fibrillation 17 and in rats with left ventricular hypertrophy. 18 Closely related to miR-1, miR-133 has two isoforms, miR-133a and miR-133b. MiR-133a is highly expressed in the heart and also plays a role in cardiogenesis and cardiac conductance. 13 It has antiapoptotic properties in cardiomyocytes 19 and represses myocardial fibrosis. 20 Similarly to miR-1, its expression was also found to be decreased in cardiac hypertrophy. 21 Another heart-enriched microRNAs belong to the miR-208 family, which consists of miR-208a, encoded by α-myosin heavy chain (MHC) gene-Myh6; miR-208b, co-expressed with β-MHC gene-Myh7; and miR-499, encoded in the Myh7b gene. In adult rodents, miR-208a is expressed exclusively in the cardiac muscle, thus cardio-specific, whereas miR-499 is relatively highly expressed in the heart and miR-208b is absent in this organ under normal conditions. α-MHC is also the predominant form of MHC, accounting for more than 90% of total expression. 22 However, in larger mammals and human beings, the major cardiac isoform is β-MHC. 23 During pathological hypertrophy, the distribution of myosin isoform changes with induction of β-MHC at the expense of α-MHC. 24 MiR-208a regulates the expression of Myh7b/miR-499 and is also needed for the up-regulation of Myh7/miR-208b and β-MHC in pathological setting. This microRNA is therefore considered to govern myofiber diversification, stress responsiveness of the heart and cardiac remodelling 22,25 and it is also important for proper cardiac conduction. 26 MiR-499, seeming to be a downstream mediator of miR-208a, 22 has antiapoptotic and proliferative properties during the late stages of cardiac differentiation. 27 Even though miR-214 was initially associated with cellular death and is dysregulated in various malignancies, it is also expressed in cardiac tissue having cardiac pathology-related influence, influencing tissue remodelling, ischaemic conditions, etc 28 This microRNA was investigated in connection with cardiac hypertrophy and fibrosis; nevertheless, the studies suggest controversial conclusions, because some indicate that miR-214 has pro-hypertrophic 29 and pro-fibrotic 30 properties, whereas others showed its protective effects in these aspects. 31,32 Based on a close relation of the above-mentioned miRNAs to cardiac injury and remodelling, we suppose that these microRNAs could also have an impact on the overwhelmed right ventricle in experimental PH and they could possibly help clarify pathological mechanisms occurring in this organ.

| Animal experiment design
In the experiment, 12-week-old male Wistar rats weighing 235-300 g (Department of Toxicology and Laboratory Animals breeding, Dobrá Voda, Slovak Republic) were randomized into two groups, depending on the treatment (either saline-control group or monocrotaline-MCT group). Monocrotaline was administered subcutaneously at the dose of 60 mg/kg in MCT group 7 whereas the control group received a corresponding volume of saline. Rats were kept under standard conditions, with access to food and water ad libitum. The rats were planned to be killed after 4 weeks since the treatment administration; however, in certain animals from the MCT group, the terminal stage of PH developed more rapidly and they had to be killed prematurely, since their survival till the 28th day after MCT administration was not expected. These prematurely terminated MCT rats (ptMCT) were observed and selected by two independent examiners, the necessary criteria being loss of

| Vital functions measurements
For haemoglobin oxygen saturation, heart and breath rate measurement, a pulse oximeter (MouseOx Plus; Starr Life Sciences, Oakmont, PA, USA) was used 24 hours before sacrifice, 6 or, in case of ptMCT group, shortly before sacrifice. A sensor collar of appropriate size was placed around the neck of conscious rats, and the vital functions of the animals were recorded and analysed accordingly to the manufacturer's instructions.

| Sample collection
Lungs and heart ventricles were harvested, blotted dry and weighted.
The ratio of RV mass to left ventricular mass plus interventricular septum (Fulton index) was used as a measure of RV hypertrophy. 8 Tissue samples of right ventricles were frozen in liquid nitrogen and stored at −80°C until further processing.

| Messenger RNA and microRNA tissue expression measurement
Both mRNA expression of cardiac failure-related indicators, such as brain natriuretic peptide (BNP)-encoding gene (Nppb), 33

| RESULTS
In rats following MCT administration, the development of PH was demonstrated by increased RV mass (including Fulton index), heart rate, lung weight and depressed oxygen saturation ( Note: Bodyweight (BW), absolute and relative mass of organs, Fulton index (right ventricular mass/ left ventricular plus septum mass), haemoglobin oxygen saturation, heart and breath rate of the rats; n(control) = 17, n(MCT) = 20, n(ptMCT) = 10; expressed as mean ± SEM, *P < 0.05 vs control group, # P < 0.05 vs MCT.

TA B L E 3
Basic characteristics of experimental groups exception of heart rate and oxygen saturation, all relevant characteristics were significantly elevated in ptMCT rats when compared to surviving MCT animals. Additionally, ptMCT rats exhibited also markedly elevated breath rate.

| Changes in the gene expression of RV impairment markers
We noted a significant eightfold increase in the expression of BNP encoding gene (Nppb) in MCT group and a 15-fold increase of Nppb in ptMCT when compared to control group (Figure 1). Gene expression of Myh6 was significantly decreased in MCT group (by 33%); this was aggravated in ptMCT group (by 88%) vs the control group.
In contrast, the Myh7 gene was up-regulated according to a similar pattern, that is with more considerable change in ptMCT (Figure 1).

| Dysregulated expression of myomiRs and miR-214 in right ventricle
We found significantly suppressed RV tissue levels of all tested my-omiRs exclusively in the ptMCT group (when compared to control group, in case of miR-1 and miR-133a also compared to MCT, see

| Expression of microRNAs significantly correlates with Fulton index
Further analyses showed a significant negative correlation between the expression of miR-1, miR-133a, miR-208a and miR-499 and Fulton index in the MCT group ( Figure 4). In contrary, there was also a significant positive correlation between miR-214 and Fulton index.

| D ISCUSS I ON
In this study, we observed significant alterations of candidate micro-RNAs in the right ventricles of monocrotaline-induced model of PH in rats in line with disease severity. The right ventricle is an essential point of interest in PAH as it is the major determinant of functional state and prognosis of the disease. 38 We focused on heart-relevant microRNAs, that is miR-1, miR-133a, miR-208a, miR-499 and miR-214 due to their high expression in the myocardium and their vital roles in cardiac development and function, and an increasing evidence about their roles in cardiovascular pathologies.
We discriminated between two groups of rats suffering from MCT-induced PH according to their disease progression. As ex- MyomiRs are intensively studied in diverse models of cardiac diseases, 13 including those of PH. 39,42,43 Although MCT administration is the most common in PH research, the data on myomiRs in right ventricle of this particular model are from scarce to non-existent, with the exception of miR-208. 39 Here, we describe a con- seen in other experimental cardiomyopathies. 12 Interestingly, miR-208a/b and miR-499 quantities were increased in left ventricular heart failure, 48 suggesting a differential expression pattern in various cardiac compartments. 49 MiR-1 regulates the expression of genes relevant in cardiac conduction and contraction. 13 The down-regulation we observed was also noted in the murine model of pulmonary artery constriction. 43 Contradictory to our observation, Reddy and colleagues reported the down-regulation of miR-1 in RV as soon as 2 days after pulmonary artery constriction which they described as early compensated hypertrophy. This down-regulation persisted until progression to decompensated cardiac hypertrophy and failure, in line with our findings. 43 Altered miR-1 levels lead to conductance defects of heart. 50,51 In both human PAH and MCT-induced PH, a proarrhythmic state is a vital contributor to morbidity and mortality. 52,53 The decrease in miR-1 levels could contribute to this condition.
With regard to miR-133a that is also a regulator of cardiac electric remodelling and exhibits antifibrotic and antihypertrophic effects in heart, 54,55 we observed a marked decrease only in ptMCT. Drake and colleagues found down-regulation of miR-133a only in models of RV failure (using Sugen5416/hypoxia and pulmonary artery banding with a Cu 2+ -depleted diet), whereas in the models of compensated RV hypertrophy (hypoxia and pulmonary artery banding), they observed no significant changes. 42 This is perfectly in accordance with our results, that is the more pronounced RV damage, the lower the miR-133a expression.
So far, miR-214 has been linked to different effects in studies concerning cardiac hypertrophy and fibrosis. [29][30][31][32] Similarly to our findings in ptMCT rats, miR-214 was found to be up-regulated in the right ventricle of the Sugen5416/hypoxia PH model 56 that is considered a model of severe PH with RV failure. 42 Reddy and colleagues also reported increased miR-214 expression in pulmonary artery banding model of RV hypertrophy and failure and associated this mi-croRNA with myocyte survival and antiapoptotic properties. 43 MiR-214 null male mice exposed to Sugen5416 and hypoxia had more pronounced RV hypertrophy when compared to their respective wild-type controls. Interestingly, this was not associated with elevation in RV systolic pressure. 56  To sum up, alterations in RV tissue levels of candidate microR-NAs reflected the worsened clinical status of rats and the more pronounced molecular indicators of cardiac damage in MCT-induced PH. We showed decreased RV levels of cardio-specific miR-208a as well as other myomiRs (miR-1, miR-133a and miR-499), whereas the expression of cardiac damage-related miR-214 was enhanced with disease severity. Further studies should focus on their particular downstream targets in preclinical research and drug development.
In conclusion, our data support the validity of concept of influential microRNAs in PH-related RV damage inspiring their further investigation as potential aid for differential diagnosis of human disease.

| CLINIC AL IMPLIC ATIONS
In this study, we showed the decreased levels of myomiRs and increased expression of miR-214 at the terminal stage of experimental PH. As these microRNAs were demonstrated to be fundamental regulators also in human cardiac pathology, 14,57 they might be in the spotlight of clinical research when searching for candidate molecules responsible for sudden worsening of RV performance in human PAH patients.

ACK N OWLED G EM ENTS
The authors gratefully acknowledge the excellent technical assis-

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
ZK, KLZ and JV performed the research; ZK, LBP, GD and EM analysed the data and devised the methodology; ZK, JK and PK wrote the paper; LP revised the manuscript; JK and PK designed the research study and supervised the work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.