Hasegawa - Journal of Pharmacology and Experimental ...jpet.aspetjournals.org/content/jpet/early/2016/03/29/jpet.115.231696.full.pdf · JPET #231696 2 Running title page: a) CNP Improves

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C-Type Natriuretic Peptide Improves Left Ventricular Functional Performance at Rest

and Restores Normal Exercise Responses after Heart Failure

Tiankai Li

Heng-Jie Cheng

Nobuyuki Ohte

Hiroshi Hasegawa

Atsushi Morimoto

David M. Herrington William C. Little

Weimin Li

Che Ping Cheng

The primary laboratory of origin: Wake Forest School of Medicine

Winston-Salem, NC (H.J.C., N.O., H.H., A.M., D.M.H., W.C.L., C.P.C.)

Affiliations: The First Affiliated Hospital of Harbin Medical University,

Harbin, China (T.L., H.J.C, W.L., C.P.C)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 29, 2016 as DOI: 10.1124/jpet.115.231696

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Running title page:

a) CNP Improves LV Performance at Rest and Exercise after HF

b) Address correspondence to:

Che Ping Cheng, MD, PhD, Section on Cardiovascular Medicine, Wake Forest School of Medicine,

Medical Center Boulevard, Winston-Salem, North Carolina 27157-1045. Phone: 336-716-2887. Fax:

336-716-5324. E-mail: [email protected] or

Weimin Li, MD, Department of Cardiology, The First Affiliated Hospital of Harbin Medical

University, NO.23 Youzheng Street, Harbin, China 150001. Phone: 86-138-0460-1998 Fax: 86-451-

367-0428. E-mail: [email protected]

c) The number of text pages: 31

The number of tables: 2

The number of figures: 4

The number of references: 71

The number of words in the Abstract: 249

The number of words in the Introduction: 684

The number of words in the Discussion: 1598

d) A list of nonstandard abbreviations used in the paper.

CNP, C-type natriuretic peptide; HF, heart failure; LV, left ventricular; LA, left atrial

e) A recommended section assignment to guide the listing in the table of contents. Section options are:

Cardiovascular


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Abstract

In heart failure (HF), the impaired left ventricular (LV) arterial coupling and diastolic dysfunction present

at rest are exacerbated during exercise. C-type natriuretic peptide (CNP) is elevated in HF. However, its

functional effects are unclear. We tested the hypotheses that CNP with vasodilating, natriuretic, and

positive inotropic and lusitropic actions may prevent this abnormal exercise response after HF. We

determined the effects of CNP (2 μg/kg plus 0.4 μg/kg/min, iv, 20 min) on plasma levels of cGMP before

and after HF and assessed LV dynamics during exercise in 10 chronically-instrumented dogs with pacing-

induced HF. We found that compared with before HF, CNP infusion caused significantly greater

increases in cGMP levels after HF. After HF, at rest, CNP administration significantly reduced LV end-

systolic pressure (PES), arterial elastance (EA) and end-diastolic pressure. The peak mitral flow (dV/dtmax)

was also increased due to decreased minimum LVP (LVPmin) and the time constant of LV relaxation (τ)

(p<0.05). In addition, LV contractility (EES) was increased. The LV-arterial coupling (EES/EA) was

improved. The beneficial effects persisted during exercise. Compared with exercise in HF preparation,

treatment with CNP caused significantly less increases in PES, but significantly decreased τ (34.2 vs 42.6

ms) and LVPmin with further augmented dV/dtmax. Both EES, EES/EA (0.87 vs 0.32) were increased. LV

mechanical efficiency improved from 0.38 to 0.57 (p<0.05). After HF, exogenous CNP produces arterial

vasodilatation and augments LV contraction, relaxation, diastolic filling and LV arterial coupling, thus

improving LV performance at rest and restoring normal exercise responses after HF.


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Introduction

Heart failure (HF) occurs when the cardiac output is unable to meet the body’s needs without an

elevated filling pressure. Thus, exercise intolerance is an important symptomatic manifestation of HF,

including HF patients with either reduced or preserved ejection fraction (Little and Borlaug, 2015;Santos,

et al., 2015;Dhakal, et al., 2015). Reduced exercise tolerance is the major cause of disability in HF

patients. In fact it is an independent predictor of hospital readmission and mortality in patients with HF

(Francis, et al., 2000). Despite enormous advances in the understanding and treatment of HF over the

years, it remains a serious and growing health problem, especially in older adults. Current treatment of

exercise intolerance is unsatisfactory in HF patients (Braunwald, 2013;Kitzman, et al., 2010;Ladage, et al.,

2013). There is an urgent need for new therapies to prevent and treat exercise intolerance in HF.

The limitation of exercise tolerance in HF results from both cardiac and peripheral factors. In HF

the exacerbation of diastolic dysfunction during exercise and the resulting increase in left atrial (LA)

pressure (P) contribute to exertional dyspnea. Previously, we have shown that in HF the impaired left

ventricular (LV) arterial coupling and diastolic dysfunction present at rest are exacerbated during exercise

(Cheng, et al., 1993;Little, et al., 2000). In addition, there is a reversal of the normal exercise-induced

augmentation of LV relaxation and a decrease in early diastolic LVP with a resulting increase in LAP. We

and others reported earlier that this abnormal exercise response is attributable to several factors such as

impaired intrinsic contractility, blunted inotropic responses to β-adrenoceptor agonists, enhanced

sensitivity of LV relaxation to exercise-induced increased systolic load, high levels of angiotensin II (Ang

II) and endothelin-1(Cheng, et al., 2001), impaired force-frequency relationship and a blunted peripheral

arterial vasodilator response (Ohte, et al., 2003) .

Emerging evidence supports C-type natriuretic peptide (CNP) as a new therapeutic option for the

treatment in HF (Del, 2013;Lumsden, et al., 2010). It is well known that an important component of

cardiovascular homeostasis is provided by the natriuretic peptides (NP). CNP, the third member of the NP

family produced by the endothelium and the heart, exhibit a range of actions. In addition to its well known


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vasodilating and natriuresis actions, CNP has been described to suppress sympathetic tone and the renin-

angiotensin system (RAS), inhibiting endothelin and vasopressin and improving cardiac β-adrenergic

regulation. These properties are beneficial in HF (Del, 2013;Lumsden, et al., 2010;Cheng, et al.,

2001;Corti, et al., 2001;Levin, et al., 1998). It is evident that CNP can act in both a paracrine and

endocrine fashion in many cardiovascular diseases. The biological actions of CNP are mediated through

specific receptors (mainly NPR-B) and the resulting elevation in cyclic guanosine monophosphate (cGMP)

(Wollert, et al., 2003). Importantly, cardiac production of CNP (Kalra, et al., 2003) and expression of

specific natriuretic peptide receptors, NPR-B are increased in HF (Lumsden, et al., 2010), which may

alter LV functional response to CNP and suggesting that CNP release may represent a cytoprotective

mechanism (Del, et al., 2008;Del, 2013;Kalra, et al., 2003;Lumsden, et al., 2010). The alterations of CNP-

induced cardiac response in HF remain unclearly defined. Specifically, the direct cardiac effects of CNP,

independent of the alterations in loading conditions, remain controversial (Hobbs, et al., 2004;Pierkes, et

al., 2002;Wollert, et al., 2003;Moltzau, et al., 2013;Moltzau, et al., 2014a). Moreover, although inhibiting

the degradation of NP or infusing CNP have been suggested as a possible new drug target for the

treatment of HF (Del, 2013;Lumsden, et al., 2010), no previous studies have examined exercise response

after CNP treatment in HF. Its role and mechanism on exercise performance in HF is unknown.

Accordingly, this study was undertaken to test the hypothesis that CNP may improve both LV

systolic and diastolic performance at rest and normalize exercise response in HF. We assessed the acute

effect of a clinically relevant dose of CNP (Nakamura, et al., 1994) on LV contractility, LV diastolic

filling, LV arterial coupling, and mechanical efficiency at rest, and during exercise in a conscious,

chronically instrumented dog model with pacing-induced HF (Bristow, 2000;Cheng, et al., 1996;Cheng,

et al., 2001;Little and Borlaug, 2015).

Materials and Methods

Instrumentation


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This investigation was approved by the Wake Forest School of Medicine Animal Care and Use

Committee. All experimental studies conformed to the Guide for the Care and Use of Laboratory Animals

published by the US National Institutes of Health (NIH Publication 8th edition, update 2011). Fourteen

healthy, adult (2 to 6 years old), heartworm-negative mongrel male dogs (body weight, 25-35 kg) were

instrumented to measure three LV internal dimensions, LVP, and LAP. One myocardial lead (Model 4312:

Cardiac Pacemakers, Minneapolis, MN) was implanted within the myocardium of the right ventricle (RV),

and the lead was attached to unipolar multiprogrammable pacemakers (Model 8329: Medtronic,

Minneapolis, MN) positioned under the skin in the chest. Hydraulic occluders were placed around the

venae cavae by a technique described previously (Cheng, et al., 1996;Morimoto, et al., 2004;Cheng, et al.,

2001;Cheng, et al., 1993).

Data collection

Studies were performed after full recovery from instrumentation (12 days after original surgery)

with the dogs standing and then running on a motorized treadmill (Model 1849C, Quinton Inc., Seattle,

WA) as previously described (Cheng, et al., 1993;Cheng, et al., 2001).

Experimental Protocol

Two separate experiments were conducted. The first was pilot dose-response study in subgroups

of conscious chronically-instrumented normal and HF dogs to determine the optimal dose of exogenous

CNP for the main experiment (detailed in Supplemental Material). The second (and main) experiment

(n=10) was to examine the functional significance of CNP (2 μg/kg plus 0.4 μg/kg/min, iv, 20 min) in HF.

Preparation and Determination of the Dosing Protocol of CNP

Some previous studies have examined the effects of intravenously-administered CNP in many

subjects including humans (Pham, et al., 1997) (Hunt, et al., 1994;Guo, et al., 2015;Igaki, et al., 1998)

and dogs (Clavell, et al., 1993;Stingo, et al., 1992;Morita, et al., 1992). In these studies, variable dosages


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of CNP were used either by a continuous intravenous infusion (0.01 to 0.8 μg /kg/min) or by a bolus

injection (0.94 or 5 μg/kg) alone to achieve higher plasma CNP levels in normal anesthetized dogs or in

normal man. However the past reports were inconsistent. Also because of the potential for complex

effects on myocardial systolic and diastolic function, afterload, and preload, the hemodynamic effects of

exogenous infusion of the CNP remain difficult to interpret. No studies have systematically assessed the

plasma levels of CNP, peripheral cGMP-generating capacity or myocardial and load reducing effects of

CNP in normal and after HF. The effects on myocardial function and loading conditions of clinically

relevant doses of CNP have not been well established.

Thus, to select the effective dosage of CNP for the current project, a dosing selection study was

performed in subgroups of normal and HF conscious dogs. In brief, on different days, animals randomly

received intravenous infusion of CNP for 20 minutes at doses of 0.1μg/kg/min; or received loading dose

of 2μg/kg first and then followed by incremental infusion dose of CNP at 0.1 μg/kg/min; 0.4 μg/kg/min

or 1.0μg/kg/min, respectively. We compared the effects of these four different dosages of CNP on

cardiac function and loading in the animals (See Supplemental Materials and Supplemental Table 1 for a

detailed description of the pilot dosing selection study and results). Based on the observations from the

dosing study on LV P-V relations, blood pressure and VED responses, and target plasma levels of the

compounds, we selected the effective dosage of CNP for the main experiment. Based on previous dose-

response studies in human and dogs, this regimen determined to be a sub-maximally effective dose.

CNP peptide of 300 μg (human, 22 amino acids, purchased from Bachem Americas, Inc.

Torrance, CA) was dissolved in 0.9% saline, sterilized by passage through a 0.2 µm Whatman Syringe

filter (Buckinghamshire, UK), 2 μg/kg was administered intravenously over 2 min, followed by an i.v.

infusion of 0.4 μg/kg/min for 20 minutes. As shown in Tables 1 to 2, CNP dosing profiles demonstrated

markedly altered LV functional performance and load-reducing effects. Of note, this dose of CNP

produced equally hypotensive action as we reported previously of ANP (Ohte, et al., 1999) and BNP

(Igawa, et al., 2000) in both normal and HF, but with minimal effects on heart rate. Our current selected


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dose of CNP also produced comparable plasma levels of cGMP (Fig 1) and CNP as reported in man and

dogs (Igaki, et al., 1998;Nakamura, et al., 1994;Clavell, et al., 1993;Stingo, et al., 1992).

Functional Significance of CNP in HF

Normal Rest and Induction of HF

Studies were performed after the animals had fully recovered from instrumentation. First, the

normal rest baseline studies were performed as previously described (Cheng, et al., 1993). After

completion of baseline studies, rapid RV pacing (at 220-240 beats/min) was initiated using the pacing

protocol to induce HF. After 4-5 weeks of rapid pacing, when the LV PED, during the non-pacing period,

had increased by more than 15 mmHg over the pre-pacing control level, we obtained HF data.

Effect of Exercise after HF without and with CNP

During the stable HF period, we examined the cardiac response to exercise before and after CNP

administration. Briefly, before each study, the pacemaker was turned off and the dog was allowed to

equilibrate for at least 40 minutes. Then, steady-state measurements were obtained at rest while the dogs

stood on a motorized treadmill (Model 1849C; Quinton; Seattle, WA) (Cheng, et al., 1993;Cheng, et al.,

2001).Variably loaded LV P-V loops were generated by transient occlusion of the venae cavae (VCO) as

previously described. The first HF exercise was then performed with the dogs running on the treadmill.

The treadmill speed was gradually increased every 1-2 minutes from 2.5 mph up to the maximum

tolerated level (4.5-6 mph). The animals exercised at this level until they could no longer keep up with the

treadmill. At submaximal levels of exercise, both steady-state and VCO data were obtained, and then the

treadmill was suddenly stopped. Data were acquired during 12 to 15-second periods throughout the

exercise protocol. We analyzed the data recorded during submaximal level of exercise to avoid marked

fluctuation by respiration. The total exercise time ranged from 4.5 to 8 minutes.

After dogs rested for 40 minutes (min), CNP was administered intravenously with a loading dose

of 2μg/kg followed by infusion of CNP 0.4 μg/kg/min for 20 min. Ten minutes after CNP treatment,

when the arterial pressure reached a stable level, steady-state hemodynamic data and caval occlusion data


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were collected at rest. Then treadmill exercise protocol was again performed and data were collected at

submaximal levels of exercise. We previously observed that there is no difference in the response to

exercise repeated after a 40-minute rest period (Cheng, et al., 1993;Cheng, et al., 2001). The values of

resting controls were also similar before initial exercise and with a 40-minute resting period after exercise.

Data Processing and Analysis

As previously described, LV volume, LV end-systolic pressure (LVPES)-end-systolic volume (VES)

relation and its slope (EES) and stroke work (SW)-end-diastolic volume (VED) relation and its slope (MSW)

were analyzed (Cheng, et al., 1996;Cheng, et al., 2001). Relaxation was evaluated by determining the

time constant of the isovolumic decrease of LVP (τ). LVP from the time of peak -dP/dt until mitral valve

opening was fit to the exponential equation LVP=PA exp (-t/τ) + PB, where t is time and PA, PB, and τ

are constants determined by the data. Although the decrease in isovolumic P is not exactly exponential,

the time constant, which is derived from the exponential approximation, provides an index of the rate of

LV relaxation (Gilbert and Glantz, 1989;Miyazaki, et al., 1990). In addition, τ was also calculated by the

Weiss method (mono-exponential decay model to zero asymptote). LV-arterial coupling was quantitated

as the ratio of EES to EA, determined as PES/stroke volume (SV). The LV P-V area (PVA), which

represents the total mechanical energy, was determined as the area under the PES-VES relation and systolic

P-V trajectory above the PED-VED curve. The efficiency of conversion of mechanical energy to external

work of the heart was calculated as SW/PVA (Suga, et al., 1979;Masutani and Senzaki, 2011;Little and

Cheng, 1993;Ohte, et al., 2003). Data acquisition and analysis were not blinded to group identity CNP

treatment due to the study design.

Determination of plasma CNP and cGMP concentrations

To determine the effects of CNP on plasma levels of cGMP before and after HF, venous blood

samples were collected at rest in the animals prior to HF before and after CNP infusion. Then blood

samples were obtained again after HF before and after CNP in these same animals. To further assess

whether HF alters plasma levels of CNP, in the subgroups of animals, blood samples were collected (into


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ice-chilled EDTA tubes containing aprotinin and processed) for CNP measurements in normal and HF

animals at baseline and after CNP administration. Plasma CNP and cGMP concentrations were

determined with the specific commercial radioimmunoassay (RIA) of CNP RIA kit and cyclic GMP RIA

kit (Phoenix Pharmaceuticals, Belmount, CA, USA) as previously described (Clavell, et al., 1993;Igaki, et

al., 1998;Del, et al., 2005;Brandt, et al., 1997) (Bruun, et al., 1989) by the Wake Forest University Health

Sciences Hypertension Core Laboratory.

Statistical Analysis

Statistical comparisons were made with Student’s t-test for paired observations and ANOVA with

the Bonferroni method of multiple paired comparisons as appropriate. Significance was established as P <

0.05. Data for steady state are expressed as means ± SD; values for LV P-V relations are expressed as

means ± SE.

Postmortem Evaluation

At the conclusion of the studies, the animals were killed by lethal injection of pentobarbital

sodium (100 mg/kg, iv), and the hearts were examined to confirm that instrumentation was properly

positioned.

Results

The data from the main experiment in response to the selected sub-maximally effective dose of

CNP (2μg/kg loading plus 0.4μg/kg/min infusion) are reported below. The additional pilot dosing study is

presented in supplemental data of online Table 1, four different dosages of CNP showed dose-related

elevations of plasma levels of CNP and reduced PES, but increased EES without significant changes in

heart rate in both normal and HF.

Increased Plasma levels of cGMP and CNP after CNP Administration: Normal vs HF

As exhibited in Figure 1A, at rest, plasma levels of cGMP were significantly elevated in HF

compared with levels observed prior to HF induction. Both before and after HF induction, CNP infusion


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caused similar (about 2.5-fold) increases in plasma cGMP levels (before HF: 7.2 to 24.7 pmol/ml; after:

20.2 to 71.6 pmol/ml). However, in response to the same dose of CNP administration, the absolute

increases of cGMP concentrations from baselines were significantly greater in HF (~3-fold higher) than

that occurred prior to HF (Δ cGMP = 51.4 vs 17.5 pmol/ml), suggesting an enhanced response in HF. In

the subgroup animals, compared with normal, after HF not only the basal concentrations of circulating

CNP were significantly higher, the same dose of CNP administration also caused greater increases in

plasma levels of CNP (Normal: 1132.6 vs 4.8 pg/ml; HF: 1984.8 vs 19.6 pg/ml) (supplemental data of

online Table 1).

Abnormal Hemodynamic Responses in Pacing-Induced HF: Rest vs Exercise

At rest, consistent with our past reports (Cheng, et al., 2001), chronic RV rapid pacing in a canine model

produced progressive LV systolic and diastolic dysfunction. As summarized in Tables 1 and 2, compared with

normal rest, LVPED, VED, VES and EA significantly increased; whereas, dP/dtmax, -dP/dtmin, SV (stroke

volume), stroke work, cardiac output all significantly decreased. LV systolic dysfunction was shown by

significant reduced EES and MSW (Table 2) with reduced LV dP/dtmax. LV diastolic dysfunction was indicated by

significantly decreased maximum dV/dt (dV/dtmax) with elevated LVPmin, LVPED, and mean LAP, as well as

prolonged LV relaxation with increased time constant of LV relaxation (τ).

During exercise, as typically displayed in Figures 2-4, such abnormalities at rest were

exacerbated. Compared with HF at rest, during exercise, the heart rate, LVPES, τ (HF exercise 42.6 vs HF

rest 39.3msec), LVPED, LVPmin (25.2 vs 23.4 mmHg) and mean LAP (31.4 vs 28.1 mmHg) all

significantly increased (Figure 2 and Table 1). These changes were accompanied by a consistent

rightward and upward shift of the early diastolic portion of the LV P-V loop (Figure 2). During early

diastole, at an equivalent LV volume, the LVP was significantly higher during exercise than at rest after

HF. Compared with the HF preparation at rest, during exercise, LV contractile performance was further

impaired as indicated by markedly rightward shifts with significant decreases in the slopes of LV PES-VES

relation (EES) (3.9 vs 4.4 mmHg/ml) and LV SW-VED relation (MSW) (52.5 vs 60.2 mmHg) (Figure 2 and


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Table 2). In addition, the impaired LV arterial coupling present at rest was exacerbated during HF

exercise. Compared with HF at rest, HF exercise caused a significant decrease in EES, while EA was

relatively unchanged, resulting in decreased EES / EA ratio (0.32 vs 0.38) with reduced SW/PVA (0.38 vs

0.43) (Table 2).

CNP Improves Hemodynamic Responses in Pacing-Induced HF: Rest vs Exercise

As shown in Table 1 and Figure 4, compared with HF at rest, CNP produced no change in heart

rate, but significantly reduced PES (CNP: 93 vs Baseline:104 mmHg), arterial elastance (EA, 8.4 vs 11.7

mmHg/ml) and PED (36.3 vs 41.2 mmHg) (p<0.05). The peak mitral flow (dV/dtmax, 204 vs 158 ml/sec)

was also increased due to decreased minimum LVP (LVPmin, 17.8 vs 23.4 mmHg) and τ (35.4 vs 39.3

msec) (p<0.05).

Importantly, as presented in Table 2 and exhibited in Figures 3A, compared with HF preparations

at rest, CNP caused leftward shifts and increased slopes of P-V relations of EES (5.6 vs 4.4 mmHg/ml)

and MSW (68.5 vs 60.2 mmHg). This indicates that in conscious dogs after HF, CNP produces a direct

positive inotropic effect on LV contractile performance. The LV-arterial coupling, quantified as EES/EA,

was improved 68% (0.64 vs 0.38) (p<0.05).

During exercise after HF, treatment with CNP prevented HF exercise-induced adverse effects on

LV systolic and diastolic dysfunction. As shown in Table 1 and displayed in Figures 2-3, compared with

exercise in HF preparations, exercise after CNP treatment significantly attenuated exercise-induced

increase in PES, reversed exercise-induced abnormal increases in LVPED and mean LAP, reversed

exercise-induced abnormal increases in LVPmin,τ, and upward shift of the early diastolic portion of LV P-

V loop, and further augmented dV/dtmax. As illustrated in Figure 4, the group means data on the

differences between HF at rest and HF exercise with and without treatment of CNP clearly revealed that

this is the opposite of the response to exercise after HF. For example, compared with HF rest, HF

exercise significantly increased τ (Δ τ =3.3 msec, HF exercise: 42.6 vs HF rest: 39.3msec), LVPmin, (ΔP =

1.8 mmHg, 25.2 vs 23.4 mmHg) and mean LAP (ΔP = 3.3 mmHg: 31.4 vs 28.1mmHg)). By contrary,


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compared with HF rest, CNP HF exercise significantly decreased τ (Δ τ = -5.1 msec, CNP HF exercise:

34.2 vs HF rest: 39.3msec), LVPmin, (ΔP = -7.1 mmHg: 16.3 vs 23.4 mmHg) and mean LAP (ΔP = -2.7

mmHg, 25.4 vs 28.1 mmHg). Thus, in HF exercise, the increased dV/dtmax is mainly due to significantly

increased mean LAP; while in CNP HF exercise, the increased dV/dtmax is attributed to the enhancement

of LV relaxation with a decrease in early diastolic LV pressure.

As demonstrated in Figure 3B, compared HF exercise, CNP caused leftward shifts of PES-VES

relations with increased EES and MSW during exercise. In addition, during HF exercise with CNP

treatment, there was a reversal of abnormal HF exercise response in ventricular-vascular coupling and

cardiac mechanical efficiency. During HF exercise after treatment with CNP, HF exercise-caused

decreases in EES and EES/EA were reversed to increases in EES (6.4 vs 3.9 mmHg/ml) and EES/EA. Thus,

treatment with CNP further improved HF exercise SW/PVA by 50% from (0.38 to 0.57) (Table 2 and

Figure 4). The duration of exercise was also significantly increased (7.8 vs 5.4 minutes).

Discussion

Exercise intolerance, a hallmark of HF remains a serious and growing health problem.

Currently, there are no satisfactory therapeutic interventions for exercise intolerance in HF

patients (Braunwald, 2013;Little and Borlaug, 2015;Santos, et al., 2015;Dhakal, et al.,

2015;Ladage, et al., 2013;Kitzman, et al., 2010). Aldosterone antagonism, angiotensin-

converting-enzyme-inhibitor therapy and beta blockade treatment improves survival in patients with

HF, but failed to improve exercise capacity in recent clinical trials (Conraads, et al., 2013;Kociol RD,

2012;Jorde, et al., 2008;Narang, et al., 1996;Ladage, et al., 2013). In fact, there has been a dearth of new

developments in HF therapies in the last decade, with the exception of the recently described angiotensin

receptor-neprilysin inhibitor of LCZ696 which prevents NP degradation, while concomitantly blocking

AT1 receptor. However, its cardiovascular outcomes in HF at rest and during exercise remains to be

investigated (Langenickel and Dole, 2012;Singh and Lang, 2015;Owens, et al., 2016).


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In the current investigation, we showed, for the first time, that administration of CNP in a canine

model of HF that mimics many features of clinical HF (Spinale, et al., 1997;Bristow, 2000;Cheng, et al.,

2001;Cheng, et al., 1993) prevented the abnormal response to exercise in HF including improved LV

diastolic filling, increased LV contractility and decreased arterial elastance with an overall improvement

in LV-arterial coupling and mechanic efficiency. This effect of CNP is more effective than what we

observed previously in the exercise canine HF model by using ANG II AT1 blockade or ET-1 antagonist

alone (Cheng, et al., 2001).

How does CNP administration prevent exercise-induced exacerbation of LV systolic and diastolic

dysfunction and restore normal exercise response in HF?

The beneficial effects of CNP we observed in the current study mainly result from reduction in

the systolic load and the positive inotropic effect of CNP. The reduction in the systolic load at rest and

during exercise in HF is due to the vasodilator effect of CNP, which is mainly attributing to natriuretic

peptide effects on cGMP to an activation of a particulate guanylyl cyclase (Wright, et al., 1996). CNP

activates the NPR-B receptor to stimulate the production and release of cGMP (Lumsden, et al.,

2010;Kuhn, 2015;Pagel-Langenickel, et al., 2007). CNP is also able to induce vasorelaxation by

hyperpolarization (Barton, et al., 1998). In addition, suppression of Ang II and endothelin-1 by CNP also

importantly contributes its systolic load reduction at rest and during exercise in HF.

The mechanism on the positive inotropic action of CNP is not entirely clear. In fact, CNP has

been reported variably to have a positive inotropic effect (Pagel-Langenickel, et al., 2007;Hirose, et al.,

1998;Wollert, et al., 2003;Zhou, et al., 2009), a negative inotropic effect (Qvigstad, et al., 2010;Moltzau,

et al., 2013;Moltzau, et al., 2014a) or a biphasic(Pagel-Langenickel, et al., 2007) inotropic effects on LV

contractile performance. These disparate observations may be due to the confounding effects of

anesthesia, open-chest surgery, tissue preparations, loading conditions, species differences and the

dosages of CNP used. Indeed, although variable dosages of CNP were used in different dose-response

studies, the effects on myocardial function and loading conditions of clinically effective doses of CNP


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have not been well established. More precise contractility assessments have not been performed, CNP’s

integrated effects on LV contractility, independent of the alterations in loading conditions, both at rest and

during exercise in HF, are not known. It remains unclear whether cGMP-generating capacity or

myocardial and load reducing effects of exogenous CNP are qualitatively or quantitatively altered in HF.

Although recent studies have suggested the efficiency of exogenous CNP in the treatment of HF, only

limited data exist to establish the superiority of CNP over the other NP. It is important to understand these

effects, especially if increasing CNP is to be used as a therapeutic strategy for patients with HF.

To address these limitations, in reference with available past dose-response studies in dogs

(Clavell, et al., 1993;Stingo, et al., 1992;Morita, et al., 1992) and man (Guo, et al., 2015;Pham, et al.,

1997;Igaki, et al., 1998;Hunt, et al., 1994), we performed dosing-response study. For the first time, we

combined both a bolus injection and continuous intravenous infusion of CNP and established effective

and stable pharmacological plasma concentrations of CNP. We simultaneously assessed cardiovascular

functional performance and plasma levels of CNP and cGMP and selected the sub-maximally effective

dose of CNP for the current study.

In the current study, we evaluated LV contractile performance after the effective dose of CNP in

conscious HF dogs with pressure-volume analysis, a load-independent measure of LV contractility as we

previously described (Little and Cheng, 1993;Cheng, et al., 1996). Compared with HF preparation, we

found that CNP caused significant increases in EES and MSW (the load insensitive index of contractility)

both at rest and during exercise, demonstrating positive inotropic effects of CNP in HF. These

observations are supported with the findings from previous studies suggesting CNP stimulation produced

positive inotropic responses may due to the direct myocardial effects (Wollert, et al., 2003;Hirose, et al.,

1998).

CNP produced an enhanced positive inotropic effect in HF in contrast to the cardiac response to

atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) observed by us (Ohte, et al.,

1999;Igawa, et al., 2000) and by others (McCall and Fried, 1990;Tajima, et al., 1998;Tsutamoto, et al.,


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1997;Nakamura, et al., 1998;Moe, et al., 1990). Previously, we reported that ANP has negative effects on

LV contractility and relaxation (Ohte, et al., 1999) whereas BNP has no direct cardiac inotropic action

both before and after HF.

Specifically, in our past studies, we first assessed the effect of similar intravenous dosages of

ANP (2 μg/kg loading dose plus 0.1 μg/kg/min infusion for 15 minutes) on LV systolic and diastolic

performance before and after HF at rest. In addition, data were collected with higher infusion rates of

ANP (0.5 and 1.0 μg/kg/min). We found that ANP produced arterial vasodilation with significantly

decreased LV PES (-9 to -10 mmHg and a load-independent depression of LV contractile function (with

about 9% reductions of EES and MSW) and slowed relaxation both before and after HF. Importantly, the

vasodilatory and cardiodepressant effects of ANP were not attenuated in HF. However, the contractile

depression and slowing of relaxation after HF are more than offset by ANP’s arterial vasodilation so that

steady-state SV, relaxation, and early diastolic function are enhanced. In another series of experiments,

we assessed the functional effects of clinically relevant doses of nesiritide (generic name of human BNP,

2 μg/kg plus 0.04 μg/kg/min, iv. 20 min) in the same canine model with pacing induced HF at rest and

during exercise (Igawa, et al., 2000). We found that after HF, at matched levels of exercise, treatment

with BNP prevented exercise-induced increases in PES, mean LAP and LVPmin. With BNP, there were no

significant changes in EES, but EES/EA was improved due to decrease in EA. τ was much shortened and

peak mitral flow was further augmented. In contrast, during HF exercise, equal hypotensive CNP was

more effective than BNP, producing decreases in LV Pmin, VES, and τ. Exercise duration in HF was

increased only by CNP.

In agreement with the observed positive inotropic effect of CNP in HF in the canine model, we

also observed positive CNP-induced modulation on LV and myocyte functional performance in both

normal and HF rats. In rats with HF, CNP caused greater improvement of intact LV and myocyte

contraction, and relaxation with further augmentation of increased [Ca2+]i transient and L-type Ca2+

current (Zhou, et al., 2009). The enhanced CNP positive modulation on cardiac performance in HF we


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observed may be due to the fact that the cardiac expression of NPR-B are increased in HF. Additionally, it

has been shown that the activity of CNP is enhanced in the absence of endogenous NO production,

indicating that CNP may play a compensatory role in protecting the heart and vasculature when NO

signaling is impaired (NO signaling dysfunction is characteristic of HF) (Lumsden, et al., 2010).

Compared with normal, after HF not only the basal concentrations of circulating CNP were

significantly higher, the same dose of CNP administration also caused greater increases in plasma levels

of CNP. Clinical pharmacokinetic studies in HF reported that the main changes in drug pharmacokinetics

seen in HF are a reduction in the volume of distribution and impairment of clearance (Shammas and

Dickstein, 1988). It is likely that factors contribute to this alteration may be involved reduced metabolism

of CNP or reduced volume of distribution in HF (Shammas and Dickstein, 1988). This new finding is

consistent with the observations made in HF patients (Del, et al., 2005). It has been demonstrated that

plasma level of CNP in healthy subjects was 2.7 pg/ml and significantly increased in HF, as a function of

clinical severity to 7.0 pg/ml, 9.6 pg/ml and 11.8 pg/ml in NYHA class II, III and IV patients,

respectively.

Past studies reported that the baseline plasma cyclic GMP concentration was higher in the HF

patients but further increases in plasma cGMP in response to ANP was limited (Moe, et al., 1992). In our

conscious dogs, compared with baselines, BNP administration produced similar increases in plasma

cGMP levels both before (135 vs 13 pmol/ml) and after HF (167 vs 23 pmol/ml) (Igawa, et al., 2000). As

opposed to ANP and BNP, in the current study, the ability of CNP to generate the second messenger

cGMP in HF was enhanced. Compared with prior to HF, CNP caused significant increases of plasma

levels of cGMP after HF, although we could not determine myocardial levels of cGMP. The cGMP

signaling plays an important role that counters a broad array of acute and chronic cardiac stress responses,

including those from beta-adrenergic stimulation, ischemic injury, and pressure and volume overload

(Kass, 2012). CNP has been reported to exert marked cGMP-mediated positive inotropic and lusitropic

effects (Lumsden, et al., 2010). The cGMP generated by NPR-B has been reported to increase β1-


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adrenoceptor-mediated positive inotropic responses through inhibition of PDE3 (Moltzau, et al., 2013). In

HF, it is possible that cGMP may affect cAMP signaling via cross-talk regulation by cGMP-regulated the

subtypes of PDEs (PDE2 or PDE3) (Takimoto, 2012;Moltzau, et al., 2014b), which leads to an increase

of intracellular cAMP and increased contractility. However, whether and to what extent administration of

CNP affects the cGMP/cAMP pathway, improving β-adrenergic stimulation hereby contributing to the

beneficial action of CNP in HF is unknown.

Of equal importance, CNP has been viewed as endogenous inhibitor of RAS and endothelin

(Sherwood, et al., 2011). Previously we reported that in HF, Ang II and endothelin-1 produce direct

depressions in intact LV contraction and exacerbate myocyte contractile dysfunction (Cheng, et al.,

1996;Suzuki, et al., 1998). We further demonstrated that in HF, circulating Ang II and endothelin-1

increase to very high levels during exercise and exacerbate the diastolic dysfunction present at rest. Thus,

inhibiting Ang II and endothelin-1 may also contribute to the increased LV contractility, relaxation and

improved LV diastolic filling at rest and during exercise after CNP administration (Cheng, et al., 2001).

Previous observations in our laboratory have demonstrated that normally-functioning LV and

arterial system are nearly optimally coupled to produce stroke work (SW) both at rest and during exercise

(Little and Cheng, 1993). In the current study, we found that during the development of HF, the EES/EA

ratio was reduced, resulting in less than maximal SW. Furthermore, this coupling ratio was further

depressed during exercise, thus contributing to exercise intolerance in HF. Treatment with CNP

significantly increased the EES/EA ratio with resulting near maximum SW both at rest and during exercise

after HF. Thus with CNP, LV mechanical efficiency was significantly augmented in HF both at rest and

during exercise. This finding may be consistent with recent views that NP has emerged as key regulators

of energy usage and metabolism, promoting lipolysis, lipid oxidation, and mitochondrial respiration

(Kuhn, 2015).

The upward shift in the early diastolic portion of the LV P-V loop that we observed during HF

exercise is similar to that reported by Miyazaki (Miyazaki, et al., 1990) in exercising dogs with coronary

stenosis as well as that found in clinical studies of exercise-induced ischemia (Tebbe, et al., 1987). In


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these studies, the decrease in LV distensibility during exercise was due to the effect of myocardial

ischemia. Although our animals did not have coronary stenosis, exercise-induced ischemia may have

contributed to our findings. Thus, CNP caused coronary vasodilatation (Hobbs, et al., 2004) may have

contributed to improved LV relaxation and LV filling during exercise after HF.

Study limitations

Several methodological issues should be considered in the interpretation of our data. First, our

observations were obtained from a pacing-induced HF canine model. The conscious dog model is a useful

model to assess drug efficacy and safety and rapid pacing produces an animal model of HF that closely

mimics that of clinical congestive cardiomyopathy including biventricular chamber dilatation with

increased LV and RV filling pressures and striking abnormalities in systolic and diastolic function

(Bristow, 2000;Cheng, et al., 1996;Spinale, et al., 1997;Little and Borlaug, 2015). However, we cannot be

certain that these results are applicable to HF from other causes such as hypertrophic cardiomyopathy.

Second, we studied the acute effects of CNP treatment. We do not know the effects of prolonged

treatment with CNP. Third, we did not examine the effects of CNP on LV end-diastolic pressure-volume

relation during exercise in this investigation. Further studies are needed to focus on this point in HF.

Fourth, we measured the plasma levels of cGMP, but we did not measure the cGMP or cAMP levels in

the heart. It is possible that its beneficial actions are attributable to the alteration on β-adrenergic

stimulation activated by subtypes PDEs regulated CNP-activated cGMP/ Protein Kinase G (PKG)

pathway (Moltzau, et al., 2014b;Moltzau, et al., 2013). Finally, the angiotensin receptor-neprilysin

inhibitor of LCZ696 has demonstrated greater efficacy than enalapril in a phase III trial in HF with

reduced ejection fraction (Langenickel and Dole, 2012;Singh and Lang, 2015). We speculate that its

ability to augment the endogenous CNP may play a major role in its greater efficacy in HF. Clearly, more

insight will be gained from our ongoing work designed to assess the influence of LCZ696 on endogenous

CNP levels as well as on cardiac performance at rest and during exercise in our canine HF model.


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In conclusion, in dogs with pacing-induced HF, the generation of cGMP in response to CNP is

not blunted. A clinically relevant dose of CNP produces arterial vasodilatation and augments LV

contraction, relaxation, diastolic filling, LV arterial coupling and mechanical efficiency; thus improving

LV performance both at rest and during exercise. This study is important in that it supports the view that

HF is a state of functional natriuretic peptide hormone deficiency (Kuhn, 2015), illuminates a new

potential mechanism of exercise intolerance in HF and points to a new molecular target for this serious

and increasing health problem.

Acknowledgements

We gratefully acknowledge the computer programming of Ping Tan; the technical assistance of Xiaowei

Zhang; and the administrative support of Stacey Belton. The authors thank Bridget Brosnihan for

performing the biochemical CNP analyses.

Authorship Contributions

Participated in research design: Li, and Cheng.

Conducted experiments: H. J. Cheng, Ohte, Hasegawa, Morimoto, and Cheng

Performed data analysis: Li, Ohte, Hasegawa, Morimoto and Cheng

Wrote or contributed to the writing of the manuscript: Li., Herrington, Little, W Li, and Cheng


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Footnotes:

a) This study was supported, in part, by grants from the National Institutes of Health (AG049770)

(H.J. Cheng); the National Institutes of Health (HL074318), American Heart Association Grant-

in-Aid (11GRNT7240020) (C.P. Cheng); and National Natural Science Foundation of China

(81270252) (W.M. Li).

b) Dr. William C. Little is deceased. We would like to dedicate this paper to the memory of Dr.

Little (5/1/1950 - 7/9/2015), and he will still be listed as a co-author.

c) This work was presented as an abstract at American Heart Association Meeting in 2014, C-

Type Natriuretic Peptide Improves Left Ventricular Systolic and Diastolic Functional

Performance at Rest and During Exercise after Heart Failure. Circulation. 2014;130:A12583

d) Send reprint requests to: Che Ping Cheng, Section on Cardiovascular Medicine, Department of

Internal Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem,

NC 27157-1045. E-mail: [email protected]


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FIGURE LEGENDS

Fig.1. Group mean data (means ± SD) (n= 10/group) of the effects of c-type natriuretic peptide (CNP)

administration on plasma levels of cGMP at rest before and after HF. The resting values of cGMP

significantly elevated after the development of HF; after CNP administration, values were further

increased to very high levels both before and after HF. *P<0.05, HF baseline vs. normal baseline;

**P<0.05, CNP vs. corresponding baselines.

Fig.2. Examples of the effects of CNP on LV diastolic filling during exercise after HF. The steady-state

LV P-V loops obtained from one animal after HF at rest and during exercise with and without the

treatment of CNP. Each loop was generated by averaging the data obtained during a 12 to 15-second

recording period, spanning several respiratory cycles. After HF, the early diastolic portion of LV P-V

loop was shifted upward during exercise, so that the early diastolic LVP was increased during exercise

after HF. After treatment with CNP, exercise produced a greater stroke volume and the abnormal exercise

response was restored to the normal downward shift during exercise, so that early diastolic LVP did not

increase, but decreased with exercise after HF.

Fig.3. Examples of the effects of CNP on LV PES-VES relations during exercise after HF. LV P-V loops

and P-V relations determined from a conscious dog after HF before and after administration of CNP.

Treatment with CNP produced leftward shifts of the LV PES-VES with increased slopes. This indicates

that CNP increased LV contractility after HF. LV P-V loops recorded following transient caval occlusions

in one conscious dog after HF at rest before and after CNP (3A) and during exercise before and after CNP

(3B). After HF, compared with rest, exercise caused a decrease in the slope of the LV PES-VES relation,

EES. Compared with HF exercise without treatment, HF exercise following treatment with CNP produced

marked leftward shifts of the LV PES-VES relation with increase in the slope of EES, indicating that CNP

increased LV contractility after HF both at rest and during exercise.


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Fig.4. Group mean data on the differences between HF at rest and HF exercise with and without treatment

of CNP on LV filling, LV arterial coupling, and stroke work (SW)/P-V area (PVA) (SW/PVA).

Compared to HF at rest, HF exercise caused an increase in dV/dtmax due to significant increases in mean

LAP, while τ and LVPmin were also significantly increased. In contrast, during HF exercise with treatment

of CNP, there was a greater increase in dV/dtmax with decreases in τ and mean LAP. During HF exercise,

EES/EA decreased, resulting from a significantly increased EA, but decreased EES, which lead to a reduction

in SW/PVA. In contrast, CNP reversed the HF exercise-induced abnormal responses and caused

significant increases in EES, EES/EA, and SW/PVA during HF exercise. *P<0.05, HF exercise with CNP vs.

HF exercise.


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Table 1. Effects of CNP on Steady-State Hemodynamics at Rest and During Exercise after HF

Before HF After HF

Normal Control HF Control CNP Treated

Rest (C) Rest (A1) Exercise (A2) Rest (T1) Exercise (T2)

Heart rate (beats/min) 108 ± 21 139 ± 15 172 ± 10 * 141 ± 18 162 ± 16 *

Maximum dP/dt (mmHg/sec) 2647 ± 293 1578 ± 183 § 2010 ± 214 * 1742 ± 179 † 2954 ± 147 *‡

Minimum dP/dt (mmHg/sec) -2235 ± 174 -1547 ± 109 § -1911 ± 215 * -1718 ± 112 † -2427 ± 203 *‡

Stroke Volume (ml) 14.5 ± 1.3 11.5 ± 1.8 § 12.3 ± 2.4 14.3 ± 1.4 † 16.8 ± 1.2 ‡

LV end-diastolic pressure (mmHg) 10.4 ± 2.7 41.2 ± 8.5 § 55.2 ± 8.6 * 36.3 ± 5.3 † 39.2 ± 4.6 ‡

LV end-systolic pressure (mmHg) 109 ± 21 104 ± 29 111 ± 27 93 ± 21 † 105 ± 28 *‡

Minimum LV pressure (mmHg) 1.1 ± 1.5 23.4 ± 2.8 § 25.2 ± 2.1 * 17.8 ± 2.6† 16.2 ± 2.5 ‡

Mean LA pressure (mmHg) 7.4 ± 1.2 28.1 ± 2.4 § 31.4 ± 2.3 * 25.3 ± 2.2 † 25.4 ± 2.6 ‡

LV end-diastolic volume (ml) 42.0 ± 4.7 55.3 ± 5.1 § 56.1 ± 5.8 48.9 ± 5.6† 54.5 ± 3.2 *

LV end-systolic volume (ml) 27.2 ± 4.2 42.4 ± 4.2 § 43.2 ± 4.4 35.2 ± 3.3 † 36.4 ± 2.6 ‡

Maximum dV/dt (ml/sec) 197 ± 19 158 ± 24 § 196 ± 27 * 204 ± 21 † 283 ± 25 *‡

Stroke work (mmHg•ml) 1641 ± 159 1043 ± 260 § 1354 ± 215 * 1460 ± 267 † 1972 ± 221 *‡

Cardiac output (ml/min) 1671 ± 210 1477 ± 162 § 2063 ± 183 * 1648 ± 143 † 2798 ± 215 *‡

EA (mmHg/ml) 6.9 ± 1.5 11.7 ± 1.3 § 12.1 ± 1.2 8.4 ± 1.4 † 7.5 ± 0.6 ‡

Time constant of relaxation (msec) 24.2 ± 1.3 39.3 ± 2.6 § 42.6 ± 2.8 * 35.4 ± 2.3 † 34.2 ± 2.4 ‡

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Values are means ± SD (n=10). CNP: C-type natriuretic peptide; HF: heart failure; LV: left ventricular; dP/dt: rate of rise of LV pressure; LA: left

atrial; maximum dV/dt: peak rate of mitral flow; EA: arterial elastance;

* P <0.05 HF exercise (A2) vs HF rest (A1);

† P <0.05 HF CNP rest (T1) vs HF control rest (A1);

‡ P <0.05 HF CNP exercise (T2) vs HF control exercise (A2);

§ P <0.05 HF control rest (A1) vs Normal control rest (C)

.




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Table 2. Effects of CNP on Pressure-Volume Relations at Rest and during Exercise after HF

Before HF After HF

Normal Control HF Control CNP Treated

Rest Rest Exercise Rest Exercise

EES (mmHg/ml) 6.8 ± 1.1 4.4 ± 0.3 § 3.9 ± 0.2 * 5.6 ± 0.3 † 6.4 ± 0. 3 *‡

EES/EA 0.98 ± 0.03 0.38 ± 0.04 § 0.32 ± 0.03* 0.64 ± 0.06 † 0.87 ± 0.08 *‡

MSW (mmHg) 89.7 ± 5.3 60.2 ± 3.6 § 52.5 ± 3.3* 68.5 ± 3.1 † 79.7 ± 2.6 *‡

SW/PVA 0.61 ± 0.02 0.43 ± 0.04 § 0.38 ± 0.03* 0.51 ± 0.02 † 0.57 ± 0.02 *‡

Values are means ± SE (n=10). CNP: C-type natriuretic peptide; HF: heart failure; EES: slope of linear PES-VES relation;

SW: stroke work; PVA: LV pressure-volume area; MSW: slope of SW-VED relation.

* P <0.05, HF exercise vs HF rest.

† P <0.05, CNP rest vs HF control rest.

‡ P <0.05, HF CNP exercise vs HF control exercise.

§ P <0.05 HF control rest vs Normal control rest.




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Hasegawa - Journal of Pharmacology and Experimental ...jpet.aspetjournals.org/content/jpet/early/2016/03/29/jpet.115.231696.full.pdf · JPET #231696 2 Running title page: a) CNP Improves