Rho‐kinase pathway activation and apoptosis in circulating leucocytes in patients with heart failure with reduced ejection fraction

Abstract Background Increased Rho‐kinase activity in circulating leucocytes is observed in heart failure with reduced ejection fraction (HFrEF). However, there is little information in HFrEF regarding other Rho‐kinase pathway components an on the relationship between Rho‐kinase and apoptosis. Here, Rho‐kinase activation levels and phosphorylation of major downstream molecules and apoptosis levels were measured for the first time both in HFrEF patients and healthy individuals. Methods Cross‐sectional study comparing HFrEF patients (n = 20) and healthy controls (n = 19). Rho‐kinase activity in circulating leucocytes (peripheral blood mononuclear cells, PBMCs) was determined by myosin light chain phosphatase 1 (MYPT1) and ezrin‐radixin‐moesin (ERM) phosphorylation. Rho‐kinase cascade proteins phosphorylation p38‐MAPK, myosin light chain‐2, JAK and JNK were also analysed along with apoptosis. Results MYPT1 and ERM phosphorylation were significantly elevated in HFrEF patients, (3.9‐ and 4.8‐fold higher than in controls, respectively). JAK phosphorylation was significantly increased by 300% over controls. Phosphorylation of downstream molecules p38‐MAPK and myosin light chain‐2 was significantly higher by 360% and 490%, respectively, while JNK phosphorylation was reduced by 60%. Catecholamine and angiotensin II levels were significantly higher in HFrEF patients, while angiotensin‐(1‐9) levels were lower. Apoptosis in circulating leucocytes was significantly increased in HFrEF patients by 2.8‐fold compared with controls and significantly correlated with Rho‐kinase activation. Conclusion Rho‐kinase pathway is activated in PMBCs from HFrEF patients despite optimal treatment, and it is closely associated with neurohormonal activation and with apoptosis. ROCK cascade inhibition might induce clinical benefits in HFrEF patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.


| INTRODUC TI ON
Increased neurohormonal drive in heart failure (HF), mainly as a result of sympathetic and renin-angiotensin system (RAS) activation, significantly contributes to pathological cardiac remodelling and disease progression. 1,2 Both neurohormonal systems promote activation of the small protein RhoA signalling pathway and its target Rho-kinase (ROCK). Activated ROCK phosphorylates and switches on several intracellular proteins, promoting cardiac hypertrophy, ventricular dysfunction, fibrosis, inflammation and apoptosis. [1][2][3][4][5] This pathway also modulates blood pressure by regulating smooth muscle contraction. 3,4 ROCK activity in circulating leucocytes has been studied extensively in patients. Elevated ROCK activity is associated with metabolic syndrome, 6 cigarette smoking and with endothelial dysfunction. 7 ROCK activity in circulating leucocytes was 90% higher in patients with cardiovascular disease compared to healthy tindividuals and the ROCK inhibitor fasudil significantly increased their forearm blood flow (but not in healthy individuals). 8 ROCK activation in peripheral blood mononuclear cells (PBMCs) has been found in human hypertension, 9,10 with higher levels in hypertensive patients with left ventricular hypertrophy (LVH). 9 In patients with heart failure and reduced ejection fraction (HFrEF), ROCK activity in circulating leucocytes is markedly elevated [11][12][13] and inversely correlated with ejection fraction. 11 There are few data in heart failure and related conditions regarding downstream effects after ROCK activation in PBMCs. In DOCA hypertensive rats, with LVH and increased myocardial fibrosis, ROCK activation in PBMCs is significantly correlated with activation of this pathway in the myocardium and with determinants of cardiac remodelling (hypertrophy, fibrosis and inflammation). 14 Most interestingly, ROCK activation increased levels of ROCK1 and the pro-inflammatory molecules p65 NF-KB, VCAM1 and IL-6 simultaneously in the myocardium and in PBMCs that decreased to control levels with the ROCK inhibitor fasudil. 14 Furthermore, in normotensive Brown Norway rats (BN), an preclinical model with genetically determined high angiotensin-converting enzyme (ACE) and angiotensin II levels, 15,16 and we previously found significantly increased phosphorylation of MYPT1, ERM and p38-MAPK as well as increased levels of p65-NF-κB at the same time in the LV and in PBMCs, and fasudil reduced them to levels observed in their respective controls. 17 Observations in rodents indicate that ROCK contributes both to myocardial fibrosis and cardiomyocyte apoptosis. 18 ROCK1 activation by caspase-3 plays an essential role in cardiomyocyte apoptosis. 19 In a transgenic mouse model of dilated cardiomyopathy, ROCK1 deletion attenuated left ventricular dilation, contractile dysfunction and cardiomyocyte apoptosis 20 and cardiomyocyte-specific ROCK1 overexpression accelerated progression to HF by increasing apoptosis and fibrosis. 21 These studies strongly suggest that ROCK activation may promote myocardial apoptosis in human HF as well.
The aforementioned observations raise several questions concerning the pathological significance of ROCK activation in HFrEF patients, specifically about the mechanisms responsible for ROCK activation, the role of downstream ROCK molecules and the clinical consequences. Moreover, there is little information about the activity of other components of this pro-remodelling pathway and regarding the relationship between ROCK activation and apoptosis in HFrEF patients.
Thus, in order to assess more comprehensively the pathophysiological role of the ROCK cascade activation in PBMCs in human HFrEF, the purpose of the study was to relate Rho-kinase activation levels with the main ROCK downstream molecules associated with myocardial remodelling as well as to apoptosis levels in circulating leucocytes in patients with HFrEF. The direct relationship of ROCK activation in the myocardium and in PBMCs was further examined in a preclinical model with activation of the renin-angiotensin system and high cardiovascular ROCK levels. 17  patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.

K E Y W O R D S
apoptosis, ERM, heart failure, MYPT1, remodelling, Rho-kinase were neoplastic disease in the last 4 years, active infection in the last 8 weeks, use of high-dose statins, 23 any other clinically significant chronic disease, obesity and diabetes.

| Echocardiographic measurements
Echocardiograms were obtained using a Philips iE33 instrument with a 2.5-MHz transducer to evaluate cardiac function at the time of blood sampling. Measurements were performed by a blinded rater according to American Society of Echocardiography recommendations. 24

| Plasma oxidative stress
Plasma malondialdehyde (MDA) and 8-isoprostane levels were measured in venous blood in both groups. MDA levels were measured by determining the content of thiobarbituric acid-reactive substances. 25
Serum levels of IL-6 and IL-8 were also determined using the Abcam ELISA kit according to the manufacturer's instructions.  26,27 were considered apoptotic. A total of 400 consecutive cells were counted in 20 sequential fields (40×). The total nuclei count and number of apoptotic nuclei were used to compute the percentage of apoptotic cells. Intra-assay and inter-assay coefficients of variation were 2.1%-3.5% and 6.2%-8.5%, respectively.

| Apoptosis levels in the myocardium and PBMCs in a preclinical model of ROCK activation
Recently, we reported a significant correlation between ROCK activation in the myocardium and in circulating leucocytes in normotensive

| Types of PBMCs involved in enhanced ROCK signalling in HFrEF patients
In order to specify in which PBMCs types ROCK is becoming ac-

| Statistical analysis
Data are presented as mean ± SD. Differences between mean values were compared using a t test, and one factor ANOVA followed by the Neuman Keuls test. Correlation analyses were performed using

| Clinical characteristics, laboratory tests and cardiac remodelling assessed by echocardiography
The aetiology of HFrEF in the patient sample was mainly dilated cardiomyopathy and coronary heart disease with standard pharmacological treatment (Table 1). Demographics, heart rate, blood pressure and blood chemistry results were similar in both groups (Table 2).
Interventricular septum thickness, left atrial diameter, LV end-diastolic diameter and pulmonary artery systolic pressure were significantly greater in the HFrEF patients, with values 13%, 29%, 40% and 61% greater than in control patients ( Table 3). The LV end-systolic diameter was 2-fold greater in the HF patients (P < .01). Systolic LV function was markedly deteriorated in the HFrEF patients.
JAK2 phosphorylation levels (upstream of Rho-kinase) were significantly increased by 3-fold in HFrEF patients as compared to controls (Table 4).

| Downstream ROCK pathway components in PBMCs (Figure 2)
HFrEF patients showed a significant 3.6-fold increase in p38-MAPK phosphorylation (P < .001, Figure 2A) and correlated with ROCK activation assessed by MYPT1-P/T levels (r = .5; P < .01). Moreover, in HFrEF patients MLC-2 phosphorylation levels were significantly increased by 4.9-fold compared with control subjects (Figure 2B), whereas JNK phosphorylation was significantly reduced by 60% in the same patients ( Figure 2C).
HFrEF patients also showed a significant 2.2, 5.8 and 7-fold increase in ICAM-1, IL-6 and IL-8 levels, respectively, as compared to control subjects (Table 4), while p65-NFkB and VCAM-1 levels were similar in the two groups.

| Circulating levels of BNP, catecholamines, angiotensins and inflammatory cytokines IL-6 and IL-8
Although the HFrEF patients were clinically stable and receiving complete pharmacological treatment, their BNP, adrenaline, noradrenaline and Ang II plasma levels were significantly increased compared with those in controls by 227%, 88%, 73% and 30%, respectively (Table 4).
Conversely, circulating levels of the vasodilatory peptide Ang-(1-9) were significantly lower by 86% in the HFrEF patients compared with control subjects ( Table 4). Levels of ROCK activation in PBMCs were directly correlated with most of circulating cathecolamines, Ang II and BNP levels and inversely correlated with angiotensin-(1-9) levels ( Figure 3).
Compared with healthy controls, HFrEF patients had a 6-fold significant increase in serum IL-6 levels and similar serum IL-8 levels (Table 4). Serum IL-6 levels were correlated with MYPT1 P/T and with ERM P/T in PBMCs (r = .45; P = .06 and r = .61; P < .01, respectively).

| Apoptosis in HFrEF patients in circulating leucocytes (Figures 4 and 5)
Patients with HFrEF displayed a significant increase in apoptosis levels determined by TUNEL in their PBMCs compared to control patients (12.3 ± 3.3% vs 4.4 ± 4.6% apoptotic nuclei, P < .001, Figure 4A). Apoptosis assessed by TUNEL was consistent with a 5.9fold increase in cleaved caspase-3 protein levels measured simultaneously in their PMBCs (P < .01, Figure 4B). Apoptosis levels in PBMCs assessed by TUNEL assay and by cleaved caspase-3 measurements were significantly correlated with ROCK activation levels measured both by MYPT1 and by ERM phosphorylation levels ( Figure 5).

| Apoptosis in the myocardium and in PBMCs in a preclinical model of ROCK activation (Figures 6 and 7)
In the preclinical model of ROCK activation in the BN rats (with genetically determined high ACE levels and ROCK activation), 15 significantly increased apoptosis levels both in PBMCs (by Tunel and cleaved caspase-3 levels) ( Figure 6A,B) and in the LV myocardium ( Figure 6C) were observed compared to the Lewis rats (with genetically determined low levels of ACE and ROCK activation). By administering the ROCK inhibitor fasudil for 7 days to BN rats, apoptosis was significantly reduced to control (Lewis rats) levels suggesting strongly a causal relationship with ROCK activation (Figure 6). In this model, a significant correlation was found between apoptotic nuclei in PBMCs and in the LV myocardium ( Figure 7A). Additionally, a significant correlation was observed between LV apoptotic nuclei with MYPT1 phosphorylation levels in PBMCs ( Figure 7B).

| Types of PBMCs involved in enhanced ROCK signalling in HFrEF patients (Figures 8 and 9)
Flow cytometry analyses in 5 consecutive HFrEF patients and controls showed that B lymphocytes (CD19+, Figure 8A,B) and monocytes (CD14+, Figure 8A,B) have low levels of MYPT1 phosphorylation compared with T lymphocytes (CD3 positive cells, Figure 9).
Two positive populations were selected for phosphorylated MYPT1, R3 and R4 in the case of CD8 T lymphocytes and R5 and R6 in the case of CD4 T lymphocytes. R3 and R5 populations displayed higher expression levels of phosphorylated MYPT1, whereas R4 and R6 populations displayed lower levels of MYPT1 phosphorylation. As shown in Figure 9, the patient's PBMCs have higher levels of MYPT1 phosphorylation compared with the control samples, which means that these two cell populations, CD4 and CD8 T lymphocytes, have a higher expression of MYPT1 phosphorylated (Figure 9).

| D ISCUSS I ON
This study examined levels of key RhoA/ROCK pro-remodelling signalling pathway molecules in PMBCs from HFrEF patients.
Significant novel findings include increased phosphorylation levels of the direct ROCK target ERM, the upstream ROCK protein JAK2,

| ROCK activation in PMBCs, cardiac remodelling and neurohormonal status in HF
Higher ROCK activation is observed in acute compared to stable HF and in patients with HF and preserved systolic function 13 and is related to mortality. 12,13 Besides, similar high ROCK activation levels  Ang II infusion in rats causes cardiac hypertrophy, which is suppressed by fasudil. 34 In DOCA hypertensive rats, fasudil reduces blood pressure and angiotensin II and increases Ang-(1-9) levels. 35 ROCK is activated by numerous factors, 1-5 and its clinical significance may involve several pathways that lead to the onset and angiotensin. [36][37][38] Thus, ROCK activity in PMBCs remained high despite optimal treatment as long as neurohormonal activation was not suppressed and myocardial remodelling not reversed.

| ROCK pathway activation in HFrEF patients
Circulating PMBCs from HFrEF patients displayed increased phosphorylation levels of the upstream ROCK cascade molecule JAK2, of two direct ROCK targets (MYPT-1 and ERM), the downstream molecules p38-MAPK and MLC-2, and higher levels of the pro-inflammatory molecules ICAM-1, Il-6 and IL-8. Reduced JNK phosphorylation levels were also found in the same cells (Table 4). Previous clinical studies in HF patients have not assessed the ROCK cascade in PBMCs but only myosin phosphatase phosphorylation. [11][12][13] Our findings here also show for the first time that in patients with HFrEF, ROCK activation in PBMCs takes place predominantly in T lymphocytes, specifically in CD4 and in CD8 T lymphocytes, which is possibly related to the higher levels of ROCK-dependent pro-inflammatory molecules ICAM-1, Il-6 and IL-8. In patients with systemic inflammatory diseases, increased ROCK activity is also observed in circulating T lymphocytes. 39 ERM phosphorylation in humans has not been assessed previously, and its pathogenic role in HF remains unknown. In rodents with PBMCs was observed previously in normotensive rats with genetically increased ACE and Ang II levels, 17 which is consistent with the concept of myocardial ROCK activation mirrored in circulating leucocytes.
Mitogen-activated protein kinases (MAPKs) participate in the development of cardiac hypertrophy, remodelling, contractile dysfunction and HF. 42 In mice, increased cardiac p38-MAPK expression is associated with reduced contractility and cardiomyopathy. 43 In rats with cardiac remodelling induced by endurance exercise, fasudil reduces cardiac hypertrophy, apoptosis, myocardial fibrosis and myocardial p38-MAPK levels. 44 Besides, a significant correlation between leucocyte p38-MAPK phosphorylation levels and cardiac and aortic wall p38-MAPK phosphorylation levels secondary to ROCK activation was found in rats. 17

| Apoptosis and ROCK activation in circulating leucocytes in HFrEF
Apoptosis is a major mechanism of HF, and increased levels of in the myocardium and in PBMCs were observed (because fasudil normalized apoptosis levels in BN rats, Figure 6). In this model, apoptosis in PBMCs and in the myocardium was correlated with ROCK activation in PBMCs (Figure 7). Even though this observation is in a different pathophysiological context, the results allow us to raise the hypothesis that apoptosis in leucocytes of HF patients is consistent with myocardial apoptosis.
Various degrees of LVH have been described in patients with dilated cardiomyopathy, and LVH in them seems to have a better prognosis than in patients without LVH. 52

F I G U R E 9
Flow cytometry of CD3 positive cells from one patient with HFrEF and a control subject. The figure shows a representative flow cytometry from a patient with HFrEF and a control patient, where the expression of phosphorylated MYPT1 was measured. The first selection was the R1 region, which represents the blood cells according to size (FSC) and cellular complexity (SSC). Then, the selection of CD3 positive cells (R2) was made, which corresponds to the population of total T lymphocytes. Subsequently, two positive populations were selected for MYPT1 phosphorylation, R3 and R4 in CD8 T lymphocytes and R5 and R6 in CD4 T lymphocytes. R3 and R5 populations are those with higher expression of phosphorylated MYPT1, whereas populations R4 and R6 are those with lower expression MYPT1 phosphorylation. As shown in the figure, the patient's blood has a higher expression of high phosphorylated MYPT1 compared with the control patient, which means that in these two cell populations, both CD4 and CD8 T lymphocytes have a higher expression of MYPT1 phosphorylated

| Study limitations
This was an observational study, without interventions performed in the HFrEF patients and a relatively small n size. However, statistical power for detecting the observed differences in the ROCK cascade and apoptosis in PBMCs was high. Another theoretical limitation in this study included a lack of analysis of the functional impact of circulating neutrophils on ROCK activity. To isolate blood cells, we used ficoll gradient, which selects PBMCs including both lymphocytes (T and B) and monocytes but not neutrophils. Neutrophils can influence T cell activation 56 and produce a marked loss of CD3+ and CD4+ T cells. 57 Thus, to determine the influence of neutrophils on ROCK activity, it is necessary to isolate lymphocytes/monocytes from neutrophils. On the other side, HF patients in the current study were clinically stable and in similar patients we previously compared ROCK activation with healthy controls and also with a second control group of hypertensive patients under pharmacological treatment and observed similar ROCK activation levels as compared to healthy controls, both control groups significantly lower compared to the HFrEF group, 11 which is consistent with subsequent findings reported by Dong et al. 12 From a clinical point of view, the current findings strongly suggest that ROCK activation determined in PMBCs may be useful to assess reverse remodelling and disease regression in this severe and highly prevalent clinical condition and could have prognostic relevance for forecasting outcome and monitoring effective treatment.
Currently, there are no ROCK inhibitors approved for clinical use in HFrEF. However, the findings provide a better understanding of the complex adverse remodelling process in the HF population and raise the possibility of therapeutic targets involving ROCK inhibition to promote reverse remodelling and clinical benefit in patients with HFrEF.

ACK N OWLED G EM ENTS
We acknowledge Ivonne Padilla for her work with the clinical samples and Roberto Gómez for his work with flow cytometry. This work was supported by grants from Fondecyt Chile (grants 1161739, 1121060 and 1150862), from Millennium Institute Chile on Immunology and Immunotherapy (grant P09/016-F) and from FONDAP Chile (grant 15130011).

CO N FLI C T O F I NTE R E S T
None.

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 request.