European Journal of Pharmacology 713 (2013) 89–93

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European Journal of Pharmacology

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Cardiovascular Pharmacology

Diminazene aceturate improves autonomic modulation in pulmonaryhypertension

Katya Rigatto a,b,n, Karina R. Casali c, Vinayak Shenoy a, Michael J. Katovich d,Mohan K. Raizada a

a Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USAb Departamento de Ciencias Basicas da Saude, UFCSPA, Porto Alegre, Brasilc Instituto de Cardiologia do Rio Grande do Sul, Porto Alegre, Brasild Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida, USA

a r t i c l e i n f o

Article history:Received 22 August 2012Received in revised form12 April 2013Accepted 18 April 2013Available online 9 May 2013

Keywords:ACE2DiminazenePulmonary hypertensionAutonomic nervous system

99/$ - see front matter & 2013 Elsevier B.V. Ax.doi.org/10.1016/j.ejphar.2013.04.017

esponding author at: Departamento de Ciencidade Federal de Ciencias da Saude de Porto Ato Alegre, RS, Brasil. Tel./fax: +55 51 3303 875ail address: krigatto@gmail.com (K. Rigatto).

a b s t r a c t

We have previously demonstrated that diminazene aceturate (DIZE), a putative angiotensin 1–7converting enzyme activator, protects rats from monocrotaline (MCT)-induced pulmonary hypertension (PH).The present study was conducted to determine if the beneficial effects of DIZE are associated withimprovements in autonomic nervous system (ANS) modulation. PH was induced in male rats by a singlesubcutaneous injection of MCT (50 mg/kg). A subset of MCT rats were treated with DIZE (15 mg/kg/day)for a period of 21 days, after which the ANS modulation was evaluated by spectral and symbolic analysisof heart rate variability (HRV). MCT administration resulted in a significant (Po0.001) increase in theright ventricular systolic pressure (62714 mmHg) when compared with other experimental groups(Control: 2676; MCT+DIZE: 3177 mmHg), while DIZE treatment was able to decrease this pressure.Furthermore MCT-treated rats had significantly reduced total power of HRV than the controls. On theother hand, although not significant, a trend towards increased HRV was observed in the MCT+DIZEgroup (Control: 108747; MCT: 1278.86 and MCT+DIZE: 40714), suggesting an improvement of thecardiac autonomic modulation. This observation was further confirmed by the low-frequency/high-frequency index of spectral analysis (Control: 0.7470.62; MCT: 1.4570.78 and MCT+DIZE: 0.3470.49)which showed that DIZE treatment was able to recover the ANS imbalance observed in the MCT-inducedpulmonary hypertensive rats. Collectively, our results demonstrate that MCT-induced PH is associatedwith a significant increase in sympathetic modulation and a decrease in HRV, which are markedlyimproved by DIZE treatment.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Pulmonary hypertension (PH) is a fatal lung disease thatnegatively affects the patient's quality of life. Endothelial dysfunc-tion, vasodilator/vasoconstrictor imbalance (Bradford et al., 2010)and disturbances in the autonomic nervous system (ANS)(Dimopoulos et al., 2009; Wensel et al., 2009) are some of theknown factors that have been linked to the pathogenesis of PH.Although considerable efforts have been directed towards under-standing the physiopathology of PH, management and cure of thisdisease still remains elusive.

Recent evidences suggest that PH may be associated withchanges in heart rate (HR) dynamics. Studies involving pulmonary

ll rights reserved.

ias Basicas da Saude,legre, Sarmento Leite,3.

hypertensive patients have shown either an increase (Sanyal andOno, 2002) or decrease (Hessel et al., 2006) in the HR mean value.On the other hand, it is widely recognized that PH is associatedwith increased sympathetic tone (Ciarka et al., 2007; Velez-Roaet al., 2004). Also, numerous studies have revealed a reducedspectral power of HR variability (HRV) (Sanyal and Ono, 2002) andincreased low-frequency (LF) versus high-frequency (HF) spectralpower ratio (LF/HF ratio) in pulmonary hypertensive subjects(Rosas-Peralta et al., 2006). These findings suggest that the cardiacANS modulation is altered in PH, resulting in an increasedsympathetic and decreased parasympathetic modulation. All ofthese changes can have profound effects on many organ systemsleading to disease severity (Gillman et al., 1993).

Clinically, ANS function can be easily evaluated by performingHR measurements in the supine position or during posturalchanges (Petrofsky et al., 2009; Robinson et al., 1966). The ECG isa low cost diagnostic tool that provides essential informationabout Q–T series (Molnar et al., 1997). Moreover, the spectral and

K. Rigatto et al. / European Journal of Pharmacology 713 (2013) 89–9390

symbolic analyses are simple methods that would allow thephysicians and researchers to collect valuable information aboutANS modulation. Measurement of the ANS function in PH patientsmay be clinically relevant, since an improvement in the ANSmodulation could potentially lower the risk factors for adversecardiovascular events (Gillman et al., 1993).

Experimental studies from our group have demonstrated thatdiminazene aceturate (DIZE) treatment significantly prevented thedevelopment of PH probably due to an increase in the vasopro-tective axis of the lung renin-angiotensin system, decreasedinflammatory cytokines, improved pulmonary vasoreactivity andenhanced cardiac functions. These beneficial effects were abol-ished by C-16, an ACE2 inhibitor. Furthermore, initiation of DIZEtreatment after the induction of PH arrested disease progression(Shenoy et al., 2013). DIZE is structurally similar to the previouslyreported angiotensin-(1–7) converting enzyme (ACE2) activatorcompound XNT, (1-[(2-dimethylamino) ethylamino]-4-(hydroxy-methyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one),but has better physicochemical characteristics. Therefore, wehypothesized is that DIZE activates ACE2 to directly or indirectlymodulate the cardiac ANS to produce its beneficial effects.

Despite the fact that autonomic nervous system imbalance is acommon finding of many diseases, its treatment is still unmen-tioned. Moreover, once the right ventricular is the major determi-nants of the functional capacity and prognosis of the PH, therelevance of evaluating the ANS function in PH and the evidencethat angiotensin converting enzyme 2 plays a key role in thephysiopathology of this disease (Ferreira et al., 2009), our goal wasto investigate whether DIZE, an ACE2 activator (Gjymishka et al.,2010), improves the autonomic modulation in the MCT-model ofPH using the spectral and symbolic analysis methods.MCT-induced PH has been extensively used as a hemodynamicallyrelevant animal model to study this disease. This model mimicsseveral key aspects of the human disorder (Werchan et al., 1989).

We propose that DIZE treatment of the pulmonary hyperten-sive rats will not only decrease the sympathetic modulation andthe sympathetic/parasympathetic ratio, but also reduce the ANSdysfunction and pulmonary pressure.

2. Materials and methods

2.1. Animals

Seven-week old male Sprague Dawley rats were housed in atemperature-controlled room (2571 1C) and were maintained ona 12:12-h light/dark cycle with free access to water and food.All procedures involving experimental animals were approved by theInstitutional Animal Care and Use Committee at the University ofFlorida and complied with National Institutes of Health guidelines.

2.2. MCT-induced pulmonary hypertension and DIZE or salinetreatment

PH was induced in two groups of rats by a single subcutaneousinjection of MCT (50 mg/kg). One group was co-treated with DIZEsubcutaneously (15 mg/kg/day) for 21 days. The other experimentalgroup was age matched control rats that were injected daily withsaline (500 ml, subcutaneously).

2.3. Cardiovascular evaluation

All the procedures and measurements were carried out onanesthetized rats. The right ventricular systolic pressure wasmeasured using a silastic catheter inserted into the right descendingjugular vein and advanced to the right ventricle, under the

influence of a rodent anesthetic cocktail comprising of ketamine(70 mg/kg), xylazine (8 mg/kg), and acepromazine (1.5 mg/kg).

The data were recorded (4000 Hz/sample rate) after stabiliza-tion of the tracing using a liquid pressure transducer, which wasinterfaced to a PowerLab (AD Instruments, Colorado Springs, CO)signal transduction unit. The appearance of a waveform was usedto confirm the positioning of the catheter in the right ventricle.Data were analyzed by using the Chart program supplied alongwith the PowerLab system.

2.4. Autonomic evaluation

After detecting the pulse intervals, the heart period wasautomatically calculated on a beat-to-beat basis as the timeinterval between two consecutive systolic peaks or pulse interval(PI). All detections were carefully checked to avoid erroneous ormissed beats. Sequences of 200–250 beats were randomly chosen(Dabire et al., 1998; Dias da Silva et al., 2006) and if they presentednon-stationary episodes, they were discarded and a new randomselection was performed. Stationarity of the series was tested aspreviously reported (Porta et al., 2004). Frequency domain analysisof HRV was performed with an autoregressive algorithm (Portaet al., 2004) on the PI interval sequences (tachogram). The powerspectral density was calculated for each time series.

In this study, two spectral components were considered: lowfrequency (LF), from 0.25 to 0.75 Hz; and high frequency (HF),from 0.75 to 3.00 Hz (Dabire et al., 1998; Dias da Silva et al., 2006;Murasato et al., 1998; Soares et al., 2004; Waki et al., 2006).The spectral components (ms2) were expressed in absolute (a) andnormalized (nu) units. Normalization consisted of dividing thepower of a given spectral component by the total power, thenmultiplying the ratio by 100. In a reduced variability condition,linear methodologies have poor applicability (Montano et al.,1994). Thus, the non-linear approach provides a new perspectivein the investigation of neural control of the cardiovascular system(Casali et al., 2008; Porta et al., 2008). Symbolic analysis isa powerful tool already validated to detect changes in autonomicmodulation of cardiovascular variability (Guzzetti et al., 2005;Porta et al., 2007) that transforms a time series into short, threebeat long patterns. The sequences are spread on six levels and allpossible patterns are divided into four groups, consisting ofpatterns with: (i) no variations (0V, three symbols equal);(ii) one variation (1V, two symbols equal and one different);(iii) two like variations (2LV); and (iv) two unlike variations(2UV) (Guzzetti et al., 2005).

2.5. Statistical analysis

The method used for statistical analysis was the non-parametric variance test of Kruskal-Wallis, complemented byDunn test. Data are presented as the mean7S.D. and Po0.05was considered statistically significant.

3. Results

Animals treated with MCT exhibited a significant increase inright ventricle systolic pressure when compared to control rats(Table 1), while rats treated with DIZE showed a decrease in thispressure. Heart rate was not different among the experimentalgroups.

MCT treatment significantly decreased the total power of HRV,HFa and LFa versus control rats. Although not significant, there wasa trend towards an increase in HFa, the parasympathetic compo-nent, when comparing MCT+DIZE with MCT group. However,DIZE treatment of MCT-treated rats resulted in a significant

Table 1Right ventricular arterial pressure (mmHg) and heart rate (bpm) values.

RVSP HR

CO (n¼7) 2676 145716MCT (n¼6) 62714a 153760MCT+DIZE (n¼6) 3177 209748

Right ventricular arterial pressure (mmHg) and heart rate (bpm) measurements.Values represent means7standard deviation; CO¼control rats; MCT¼rats treatedwith monocrotaline; MCT+DIZE¼rats treated with monocrotaline and DiminazeneAceturate. RVSP¼Right Ventricular Systolic Pressure; HR¼heart rate in beats perminute.

a po0.05 vs. CO and MCT+DIZE.

Table 2Spectral and symbolic analysis results.

CO MCT MCT+DIZE P

Spectral analysisHRV (ms2) 108747 1278.86a 40714 0.000LFa (ms2) 36726 6.175.04a 6.275.3a 0.02HFa (ms2) 59729 4.373.1a 30716 0.001LFnu 38717 55715 19720b 0.009HFnu 62717 45715 81720b 0.009LF/HF ratio 0.7470.62 1.4570.78 0.3470.49b 0.01

Symbolic analysis (%)0V pattern 5.6672.54 19714 4.0673.51b 0.021V pattern 4078.86 3778.45 27713 0.1722LV pattern 1172.78 7.1676.45 1277.92 0.1302UV pattern 4378.75 3775.02 56715 0.054

Spectral and symbolic analysis results. CO¼control rats, (n¼7); MCT¼rats treatedwith monocrotaline, (n¼6); MCT+DIZE¼rats treated with monocrotaline+Dimin-azene Aceturate, (n¼6). HRV¼Heart rate variability; LF¼Low frequency compo-nent; HF¼High frequency component; a¼absolute; nu¼normalized; Datarepresent means7standard deviation. A non-parametric Kruskal-Wallis test wasused to detect differences between groups; Data represent means7standarddeviation Po0.05 was considered statistically significant.

a vs. control.b vs. MCT.

K. Rigatto et al. / European Journal of Pharmacology 713 (2013) 89–93 91

improvement in power spectrum parameters, such as LFnu, HFnuand LF/HF ratio, versus MCT alone group. MCT-treated rats haveimpaired cardiac ANS modulation and DIZE improves the ANSbalance in favor to parasympathetic modulation. The significantand inverse proportion between HFnu and LFnu componentsobserved in MCT+DIZE rats as compared to MCT alone groupconfirm these results.

In addition, in the MCT+DIZE group there was 19% of sympa-thetic and 81% of parasympathetic modulation to the heart. In theMCT group, the proportion was 55% for sympathetic participation,and 45% for parasympathetic modulation. This observation isconsistent with LF/HF ratio, which is significantly decreased inthe MCT+DIZE group compared to MCT alone group but not fromcontrols (Table 2).

Symbolic analysis also showed that MCT induced an increase in0V pattern (%), an indication of increased percentage of sympa-thetic modulation, compared to MCT+DIZE (Table 2 and Fig. 1).

Collectively, symbolic and spectral analysis indicated that DIZEimproved the autonomic modulation to the heart.

4. Discussion

The most significant observation of this study is that chronictreatment with DIZE improves the ANS modulation in the MCT-model of PH. DIZE increased the cardiac parasympathetic anddecreased the sympathetic modulation, thus reversing the

imbalance in the ANS modulation elicited by the pulmonaryhypertensive state.

Our current findings agree with the published data showingthat MCT treatment increases the cardiac sympathetic systemparticipation (Fauchier et al., 2006; Goncalves et al., 2010;Ishikawa et al., 2009). Moreover, activation of the sympatheticnervous system has been correlated with PH severity (Wenselet al., 2009) and development of right ventricle hypertrophy inhumans (Ciarka et al., 2007; Dimopoulos et al., 2009; Folino et al.,2003; Velez-Roa et al., 2004; Wensel et al., 2009).

Our results are consistent with the previous reports involvinganimal models (Fauchier et al., 2006; Goncalves et al., 2010) andclinical subjects (Dimopoulos et al., 2009; Fauchier et al., 2004;Fauchier et al., 2006; Folino et al., 2003; Rosas-Peralta et al., 2006;Wensel et al., 2009) whereupon, a decrease in the spectral powerof HRV was observed with pulmonary hypertension.

In the current study, the MCT alone group showed a significantdecrease in the spectral power of HRV, LFa and HFa as compared tothe control group. The reduction in sympathetic and parasympa-thetic modulation observed in the MCT-treated rats has also beenreported in pulmonary hypertensive patients (Fauchier et al.,2004). Similar to our findings in the MCT group, it has been shownin the literature that PH provokes a significant reduction in bothLFa and HFa spectral power in animals (Fauchier et al., 2004) andsubjects (Wensel et al., 2009).

The decrease in total power of HRV, seen in MCT group,indicates impairment in the ability of HR to blunt the increase inarterial blood pressure. On the other hand, there was an increasein parasympathetic and a decrease in sympathetic modulation tothe heart in the MCT+DIZE group versus MCT alone. This conclu-sion is also based on the significant improvement in LF/HF ratio,seen after DIZE treatment. Likewise, similar findings have beenreported in PH patients with an increase in LF versus HF spectralpower ratio (LF/HF ratio) (Rosas-Peralta et al., 2006). The MCT+DIZE group showed a relative decrease in sympathetic modula-tion, as demonstrated by LFnu, and a corresponding increase in theparasympathetic modulation, as indicated by HFnu, comparedwith MCT rats. These findings strongly indicate that DIZE treat-ment alters the autonomic balance in favor of parasympatheticmodulation in the MCT-induced PH.

Studies have reported that DIZE evoked either a decrease(Milner et al., 1997) or no change (Joubert et al., 2003) in arterialblood pressure. A possible explanation for this reduction in arterialblood pressure was put forward by Wien (1943) who postulatedthat DIZE might induce parasympathetic modulation, by inhibitingacetyl-cholinesterase, an enzyme responsible for degrading thekey parasympathetic mediator, acetylcholine (Wien, 1943). Thismight be the case since it has been reported that large doses ofDIZE, when administered to animals, induces diarrhea and vomit-ing (Milner et al., 1997; Naude et al., 1970), classic symptoms ofparasympathetic activation. However, contradicting this hypothesiswere the findings of Milner et al. (1997) who demonstrated in dogsthat DIZE did not change the concentration of acetyl-cholinesteraseeither in the plasma or red blood cells Milner et al., 1997.

We believe that circulating acetyl-cholinesterase concentrationmay not reflect the parasympathetic nervous system modulation.As with the circulating levels of catecholamines in PH, which couldbe increased (Nagaya et al., 2000) or be within normal limits(Richards et al., 1990), the acetylcholinesterase levels may as wellvary in circulation. Furthermore, another hypothesis could be thatthe parasympathetic system modulates the sympathetic nervoussystem activity centrally.

One of the limitations of our study is that all rats were underketamine and xylazine anesthesia, which probably explains whyour results show lower HR than in conscious Sprague Dawley rats.On the other hand, all groups were under anesthesia during the

Fig. 1. An example of symbolic analysis of pulse interval (PI) series. The column shows the power spectrum of the time series: the frequency, expressed in Hz, is reported onthe x-axis, while the power spectral density of PI series, expressed in ms2, is reported on the y-axis. The data expressed in the pie-chart represent the symbolic patterndistribution of the time series: all patterns are divided into the four families of patterns, i.e., 0V, 1V, 2LV, 2UV and the percentages of occurrence of the four families areshown. The upper panel shows an example of raw data obtained by intra-ventricular measurements (A) and a pulse interval series constructed from peak detection (B).On the lower panel are MCT treated animal (C) and MCT+DIZE (D) treated rats.

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data collection, indicating that the differences found between thegroups were not due to the procedure.

The exact mechanism by which DIZE acts remains to bedetermined. We had previously demonstrated that ACE2 activationcan be a therapeutically relevant approach for treating andcontrolling PH (Ferreira et al., 2009). But, whether the beneficialeffects of DIZE on autonomic function and right ventricle pressurein PH are due to ACE2 activation and/or to acetyl-cholinesteraseinhibition are still important questions that need to be addressed.On the other hand, it is evident from our study that parasympa-thetic modulation, at least in part, is responsible for the beneficialeffects against MCT-induced PH. We did provide evidences thatDIZE treatment of PH rats alters the autonomic balance in favor ofparasympathetic modulation.

It is very well established that for every mmHg that arterialblood pressure falls there is a proportional reduction in the risk forcardiovascular events. Similarly to arterial blood pressure, the HRVdecrease may not always be significant, but its impact on patientsurvival may be very relevant.

It is also well recognized that under chronic left heart failure,the sympathetic activity is increased and blockade of the sameimproves symptoms, cardiac function and the prognosis ofpatients (Swedberg et al., 2005). Future studies need to focus onthe function of ANS in PH. Measurement of cardiac autonomic

function and its treatment may play an important role in prog-nostic risk stratification and effectively determine the clinicaloutcomes in patients suffering from PH.

5. Conclusion

Despite the efforts of the scientific community, the survival rateof patients with PH remains poor and unacceptable. Thus, a drugthat improves the ANS balance and decreases the ventriclepressure, certainly will postpone the transition from compensatedhypertrophy to maladaptive remodeling and dilatation, improvingsurvival in PH.

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