Thrombopoietin modulates cardiac contractility in vitro and contributes to myocardial depressing...

ORIGINAL CONTRIBUTION

Thrombopoietin modulates cardiac contractility in vitroand contributes to myocardial depressing activity of septicshock serum

Enrico Lupia • Tiziana Spatola • Alessandra Cuccurullo • Ornella Bosco •

Filippo Mariano • Angela Pucci • Roberta Ramella • Giuseppe Alloatti •

Giuseppe Montrucchio

Received: 23 February 2010 / Revised: 16 April 2010 / Accepted: 19 April 2010 / Published online: 14 May 2010

� Springer-Verlag 2010

Abstract Thrombopoietin (TPO) is a humoral growth

factor that has been shown to increase platelet activation in

response to several agonists. Patients with sepsis have

increased circulating TPO levels, which may enhance

platelet activation, potentially participating to the patho-

genesis of multi-organ failure. Aim of this study was to

investigate whether TPO affects myocardial contractility

and participates to depress cardiac function during sepsis.

We showed the expression of the TPO receptor c-Mpl on

myocardial cells and tissue by RT-PCR, immunofluores-

cence and western blotting. We then evaluated the effect of

TPO on the contractile function of rat papillary muscle and

isolated heart. TPO did not change myocardial contractility

in basal conditions, but, when followed by epinephrine

(EPI) stimulation, it blunted the enhancement of contractile

force induced by EPI both in papillary muscle and isolated

heart. An inhibitor of TPO prevented TPO effect on cardiac

inotropy. Treatment of papillary muscle with pharmaco-

logical inhibitors of phosphatidylinositol 3-kinase, NO

synthase, and guanilyl cyclase abolished TPO effect,

indicating NO as the final mediator. We finally studied the

role of TPO in the negative inotropic effect exerted by

human septic shock (HSS) serum and TPO cooperation

with TNF-a and IL-1b. Pre-treatment with the TPO

inhibitor prevented the decrease in contractile force

induced by HSS serum. Moreover, TPO significantly

amplified the negative inotropic effect induced by TNF-aand IL-1b in papillary muscle. In conclusion, TPO nega-

tively modulates cardiac inotropy in vitro and contributes

to the myocardial depressing activity of septic shock

serum.

Keywords Thrombopoietin � Myocardial dysfunction �Contractility � Sepsis � Nitric oxide

Introduction

Thrombopoietin (TPO) is a humoral growth factor that was

originally identified for its ability to stimulate the proli-

feration and differentiation of megakaryocytes [23, 30]. It

is constitutively produced by the liver and kidneys, and is

then cleared from circulation upon binding with its recep-

tor, c-Mpl, expressed mainly on platelets and megakaryo-

cytes [23, 30]. In addition to its action on megakaryocytes

and bone marrow progenitor cells [23, 30], TPO directly

modulates the response of mature platelets to several

stimuli and thereby their homeostatic potential [37, 38],

G. Alloatti and G. Montrucchio equally contributed.

E. Lupia � T. Spatola � A. Cuccurullo � O. Bosco �G. Montrucchio (&)

Department of Clinical Pathophysiology, University of Turin,

Via Genova 3, 10126 Turin, Italy

e-mail: [email protected]

R. Ramella � G. Alloatti

Department of Animal and Human Biology, University of Turin,

Turin, Italy

F. Mariano

Department of Medicine Area, Nephrology and Dialysis Unit,

CTO Hospital, Turin, Italy

A. Pucci

Department of Pathology, Regina Margherita Hospital,

ASO OIRM-S.Anna, Turin, Italy

Present Address:T. Spatola

Department of Medicine and Experimental Oncology

and Molecular Biotechnology Center,

University of Turin, Turin, Italy

123

Basic Res Cardiol (2010) 105:609–620

DOI 10.1007/s00395-010-0103-6

and influences the activity of white blood cells, particularly

polymorphonuclear leukocytes and monocytes [6, 50].

Elevated circulating TPO levels have been reported in

different clinical conditions, from hematological diseases

[8, 15, 24] and acute coronary syndromes [34, 44] to sepsis

[11, 22, 47]. High levels of TPO have been shown indeed

in healthy volunteers after endotoxin infusion [47], and in

children and adult patients with sepsis [11, 22]. Moreover,

it has been shown that disease severity is the major

determinant of elevated TPO levels in septic patients [52],

and that increased TPO levels may enhance platelet acti-

vation during burn injury and sepsis, potentially partici-

pating to the pathogenesis of multi-organ failure in these

diseases [35].

Myocardial dysfunction is common in patients with

sepsis and is associated with high risk to develop multi-

organ failure and high mortality rate [27, 36, 42]. Septic

cardiomyopathy is characterized by reversible biventricular

dilatation, decreased ejection fraction, and impaired

response to fluid resuscitation and catecholamine stimula-

tion [27, 36, 42]. Although intrinsic cardiac factors have

been implicated in this complex condition [27, 36, 42], the

causal role of circulating factors has been extensively

studied [28, 36, 42], following the observation that serum

from patients with septic shock decreases myocyte con-

tractile function, and that this effect correlates with the

reduction of the patient’s left ventricular ejection fraction

[39]. A pivotal role for tumor necrosis factor (TNF)-a and

interleukin (IL)-1b in mediating this depressant activity has

clearly emerged from subsequent studies [18]. Each indi-

vidual cytokine, although at supra-physiological concen-

trations, as well as the combination of the two at

concentrations similar to those measured in the blood-

stream of septic patients, is able to reproduce in vitro the

depressant effect of septic serum [25]. This response is

mainly mediated by the production of nitric oxide (NO)

and cyclic guanosine monophosphate (cGMP) [26],

although also NO-independent mechanisms have been

involved [29]. No data on the potential contribution of TPO

to myocardial depression during sepsis is currently avail-

able. Aim of this study was to investigate whether TPO

affects myocardial contractile function and contributes to

the myocardial depressing activity of septic shock serum.

Methods

Cell culture

H9C2 cardiomyocytes [20] were obtained from American

Type Culture Collection (Manassas, VA, USA). Adult rat

ventricular cardiomyocytes were obtained by enzymatic

dissociation as previously described [16].

Human heart samples

c-Mpl expression was evaluated in snap-frozen left

ventricle samples obtained at the time of cardiac trans-

plantation at the Torino Heart Center from six patients with

end-stage dilated or ischemic cardiomyopathy. The study

was conducted according to the Helsinki Declaration and it

was approved by the Institutional Ethical Committee.

Immunofluorescence and confocal microscopy studies

Indirect immunofluorescence on cultured cardiomyocytes

was performed by standard procedure using as primary

antibody an anti-c-Mpl monoclonal antibody directed

against the extracellular domain (mouse IgG2B anti-TpoR-

CD110, R&D Systems Inc., Minneapolis, MN; 10 lg/mL).

Confocal fluorometric studies were performed using a

laser scanning confocal system (Fluoview 200, Olympus

America, Melville, NY) mounted on an inverted micro-

scope (model IX70, Olympus) equipped with a 960

oil-immersion objective (NA 0.17). Cells were seeded

on glass-bottom dishes (35 9 22 mm, Willco Wells,

Amsterdam, The Netherlands) at a density of 5,000

cells/cm2.

RT-PCR

RT-PCR was performed using standard procedure. Briefly,

total RNA was extracted using TRI reagent (Sigma

Chemical Co, St. Louis, MO) according to the manufac-

turer’s directions. The final RNA pellet was dissolved in

10 lL of diethyl pyrocarbonate water and stored at -70�C;

1 lg of total RNA was reverse-transcribed using a First

Strand Synthesis Kit (Boehringer Mannheim, Indianapolis,

IN). Fixed amounts of cDNA (2 lL for c-Mpl and 2 lL

from 1/10 dilution for the glyceraldehyde-phosphate

dehydrogenase [GAPDH]) were directly used for individ-

ual PCR amplifications. Sequence-specific oligonucleotide

primers were designed (c-Mpl: forward 50 AGA ACC CAC

AGA GTG GTG TG, reverse 50 CAC CAT CCA GGA

GCA AGA AT [14]; GAPDH: forward 50 CCA CCC ATG

GCA AAT TCC ATG GCA, reverse 50 TCT AGA CGG

CAG GTC AGG TCC ACC. Times and temperatures for

denaturation, annealing and extension were 30 s 94�C, 45 s

55�C, and 1 min 72�C, respectively. Amplification prod-

ucts were visualized by ethidium bromide staining after

agarose gel electrophoresis.

Flow cytometry

c-Mpl surface expression on cardiomyocytes was examined

by flow cytometry with the indirect immunofluorescent

method using standard procedure. H9C2 cardiomyocytes

610 Basic Res Cardiol (2010) 105:609–620

123

were collected with non-enzymatic cell dissociation solu-

tion (Sigma, Saint Louis, MI, USA) and blocked with PBS-

BSA 1% for 30 min at room temperature. Cells were then

incubated with a mouse IgG2B anti-TpoR-CD110 mono-

clonal antibody (5 lg/mL; R&D Systems Inc) or the non-

immune isotypic control antibody (mouse IgG2B, 5 lg/mL;

Ancell corporation, Bayport, MN) for 30 min at 4�C. Cells

were then stained with Alexa Fluor 488-conjugated anti-

mouse IgG secondary antibody (Molecular Probes, Eugene,

OR) for 30 min at 4�C. At least 10,000 cells from each

sample were analyzed on an EPICS-XL flow cytometer

(Beckman Coulter, Hialeah, FL) and the data were pro-

cessed using EXPO32 software (Beckman Coulter).

Results were expressed as percentage of c-Mpl-positive

H9C2 cells.

Western blotting

For c-Mpl detection, protein extracts (20 lg) from cul-

tured H9C2 cardiomyocytes or human hearts were

resolved by 8% SDS/PAGE under reducing conditions,

and electroblotted to nitrocellulose membranes that were

blocked overnight in TRIS-buffered saline (TBS) con-

taining 0.1% Tween 20 and 5% bovine serum albumin.

Equivalent protein loading was ensured by Ponceau red

staining of gels after transblotting. Filters were then

reacted with an anti-c-Mpl monoclonal antibody (1:1,000;

R&D Systems Inc) for 1 h at room temperature, washed

three times, and incubated with a horseradish peroxidise

(HRP)-labeled sheep anti-mouse IgG secondary antibody

(1:25,000, Amersham Biosciences) for 1 h at room

temperature. The immunocomplexes were visualized

using ECL-immunoblotting detection kit (PerkinElmer,

Waltham, MA).

In order to study Akt1 activation in H9C2 cells and

isolated rat papillary muscles, protein extracts (50 lg)

were resolved by 10% SDS-PAGE and transferred to

nitrocellulose membranes that were blocked in TBS con-

taining 0.1% Tween 20 and 2% Blocking Agent (Amer-

sham Biosciences, Uppsala, Sweden) for 1 h at room

temperature. Filters were then incubated overnight at 4�C

with a rabbit polyclonal antibody specific for phosphory-

lated-Akt (1:1,000; Cell Signaling Technology, Danvers,

MA), and then probed with a HRP-labeled goat anti-rabbit

IgG secondary antibody (1:5,000; Pierce Biotechnology,

Rockford, IL) for 1 h at room temperature. To confirm

equal protein loading, blots were stripped and re-blotted

with a mouse polyclonal antibody specific for total Akt1

(1:5,000; Cell Signaling Technology), and then probed

with a sheep anti-mouse HRP-linked antibody (1:25,000;

Amersham Biosciences). The immunocomplexes were

visualized using ECL-immunoblotting detection kit

(PerkinElmer).

Septic shock patients

We studied six patients with septic shock, defined

accordingly to the criteria for the diagnosis of sepsis [31],

and six healthy subjects (Table 1). Exclusion criteria were

previously detailed [35].

The control group consisted of six healthy volunteers,

receiving no medications. None had shown any evidence of

febrile illness during the previous 2 weeks.

The investigation conforms with the principles outlined

in the Declaration of Helsinki and was approved by the

Institutional Ethical Committee. Informed consent was

obtained from the patients or their caring relatives.

Blood collection was performed using a central venous

catheter or, for healthy subjects, by clean venipuncture

using a 19-gauge butterfly infusion set, without venous

stasis. After discarding the first 4 mL, blood entered Vac-

utainer tubes and was centrifuged at 1,600g for 10 min at

4�C. Serum was then frozen and stored at -70�C.

Animals

Female Wistar rats (200–300 g body wt) were allowed

ad libitum access to tap water and standard rodent diet. The

investigation conforms with the Guide for the Care and

Use of Laboratory Animals published by the US National

Institutes of Health (NIH Publication No. 85-23, revised

1996), in accordance with Italian law (DL-116, 27 January

1992), and was approved by the local ethical committee.

Isolated papillary muscle and experimental protocol

Rat papillary muscles were prepared as previously descri-

bed [16]. Briefly, they were dissected free from the left

ventricle under a stereomicroscope and superfused with

oxygenated Tyrode’s solution at 36�C. Papillary muscles

were driven at constant frequency (120 beats/min) with a

pair of electrodes connected to a stimulator (302 T Ana-

pulse, W.P. Instruments, New Haven, CT) via a stimulus

isolator (model 305-R, W.P. Instruments) operating in

constant-current mode. Isometric twitches were evaluated

by a transducer (model 60-2997, Harvard Instruments) and

continuously acquired and recorded by a PowerMac com-

puter using Labview software (National Instruments). The

same software was used to measure developed peak

mechanical tension (Tmax). Before each experiment, pap-

illary muscles were equilibrated in oxygenated (100% O2)

Tyrode solution for at least 30 min. Each treatment lasted

for 20 min, then the perfusion was switched to Tyrode’s

solution alone to study the reversibility of the effects.

The effects exerted by increasing concentrations of

recombinant mouse TPO (25–500 pg/mL; R&D Systems

Inc.) on contractile force were studies both under basal

Basic Res Cardiol (2010) 105:609–620 611

123

conditions and after beta-adrenergic stimulation with epi-

nephrine (EPI, 3 lmol/L, Helena Laboratories, Beaumont,

TX). After stimulation with EPI, papillary muscles were

washed with Tyrode’s solution for 30 min to remove any

effect due to desensitization of adrenergic receptors by

prior stimulation.

To study the role of endocardial endothelium, papillary

muscles were treated with 0.5% Triton X-100 for 1–2 s,

then challenged with TPO [16].

In separate experiments, we studied the effects on the

contractile force of TNF-a (10 ng/mL) and IL-1b (10 ng/

mL), alone or in the presence of TPO (200 pg/mL), and of

serum samples (10% in Tyrode’s solution) obtained from

septic patients or healthy subjects.

In selected experiments, TPO, TNF-a, IL-1b or human

serum samples were pre-incubated with a TPO receptor

(TPOR)-Fc chimera (1.25 lg/mL; R&D Systems Inc.) for

5 min at 37�C, then the mixture of sample and TPOR-Fc

chimera was added to papillary muscles. It has been pre-

viously shown that TPOR-Fc chimera inhibits the proli-

feration induced by TPO on MO7e cells [2], as well as the

priming effect exerted by TPO in platelet-rich plasma and

whole blood [34].

Specific pharmacological inhibitors were used to evaluate

the role of the phosphatidylinositol 3-kinase (PI3K)-

Akt1-NO synthase-guanylyl cyclase pathway in the altera-

tions of cardiac response to EPI induced by TPO

(200 pg/mL). Wortmannin (100 nmol/L), 1H-[1, 2, 4]

oxidiazolo[4,3a]quinoxaline-1-one (ODQ, 10 lmol/L), and

NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), all

from Sigma Chemical Co, were used to block PI3K activity,

guanylyl cyclase activity, and NO synthesis, respectively.

The biologically inactive enantiomer of L-NAME, D-NAME

(1 mmol/L; Sigma Chemical Co.), was used as control. All

solutions containing the drugs were prepared immediately

before the experiments.

Isolated heart preparation

Rat hearts were rapidly excised, placed in ice-cold buffer

solution, weighted, and then attached to the perfusion

apparatus and retrogradely perfused at constant flow

(7.8 ± 0.3 mL/min/g) through a peristaltic pump with

oxygenated (100% O2) Tyrode’s solution. Hearts were kept

in a temperature-controlled chamber (36�C), electrically

paced at 280 beats/min through a 302 T Anapulse Stimu-

lator connected to a 305-R Stimulus Isolator (W.P.

Instruments, New Haven, CT, USA). A polyvinyl-chloride

balloon was placed into the left ventricle and connected to

an electromanometer to record left ventricular pressure

(LVP), which was visualized on a Tektronix 2211 digital

storage oscilloscope and continuously acquired and recor-

ded by a Power Mac computer, using the Labview Soft-

ware (National Instruments Corp., Texas, USA). Coronary

flow (CF) was monitored with an electronic drop counter

placed along the perfusion line. Isolated hearts were

allowed to stabilize for 30 min; at this time baseline

parameters were recorded. After stabilization, hearts were

treated with TPO (200 pg/mL) both under basal condi-

tions and after beta-adrenergic stimulation with EPI

(0.1 lmol/L). After stimulation with EPI, hearts were

washed with Tyrode’s solution for 30 min to remove any

effect due to desensitization of adrenergic receptors by

prior stimulation. In selected experiments, TPO was incu-

bated with the TPOR-Fc chimera (1.25 lg/mL) for 5 min

at 37�C before the administration.

Statistical analysis

Data represent mean ± standard error. Statistical analyses

were performed with GraphPad Prism 4.00 for Windows

(GraphPad Software, La Jolla, CA, USA) using the Stu-

dent’s t test or ANOVA followed by Newman–Keuls

Table 1 Clinical characteristics of patients with septic shock and healthy control subjects

Characteristics Healthy subjects (n = 6) Septic shock patients (n = 6) P value

Age (years) 34.60 ± 4.25 37.67 ± 18.92 0.7507

Gender (male/female) 2/4 5/1

Outcome (dead/alive) NA 3/3

Platelets (109 L-1)a 240.75 ± 32.94 197.83 ± 92.54 0.4065

Leukocytes (106 L-1)a 5,955 ± 582.32 19,350 ± 12,893 0.0293

Monocytes (106 L-1)a 435 ± 155.03 1,090 ± 702.54 0.1094

Thrombopoietin (pg/mL)a 54.42 ± 8.55 704.96 ± 458.30 0.0110

Data represent mean ± standard error. The last right column reports the P value obtained by comparing the two experimental groups using the

Student’s t test

NA non-applicablea Data at enrolment


123

multicomparison test, as appropriate. A P value of \0.05

was considered significant.

Results

Expression of thrombopoietin receptor (c-Mpl) by

cardiomyocytes and heart tissue

Cultured H9C2 cardiomyocytes express c-Mpl mRNA

and protein, as shown by RT-PCR (Fig. 1a) and western

blot (Fig. 1b). Indirect immunofluorescence experiments

using an antibody specific for the extracellular domain of

c-Mpl showed a typical surface expression of c-Mpl,

which bound to H9C2 cells fixed with ethanol with a

punctuate granular pattern (Fig. 1c–e). c-Mpl expression

by H9C2 cardiomyocytes was also showed by flow

cytometry (Fig. 1f). Finally, membrane-specific expres-

sion of c-Mpl was confirmed by confocal microscopy

(Fig. 1g).

c-Mpl expression was also evaluated in ventricular

cardiomyocytes obtained by enzymatic dissociation from

young adult rats using both indirect immunofluorescence

and flow cytometry (not shown).

Fig. 1 Identification of TPO receptor c-Mpl in myocardial cells and

tissue. a Representative PCR gels for c-Mpl and GAPDH expression

in H9C2 cardiomyocytes. Lane 1 100 bp molecular weight markers;

Lane 2 PCR for c-Mpl, no cDNA control; Lane 3 PCR for c-Mpl

(200 bp); Lane 4 PCR for GAPDH, no cDNA control; Lane 5 PCR for

GAPDH, no RT control; PCR for GAPDH (598 bp). b Western blot

analysis of c-Mpl expression in H9C2 cardiomyocytes. Lane 1 Human

platelet lysate (positive control); Lane 2 H9C2 cell lysate. c–e Indirect

immunofluorescence staining for c-Mpl in H9C2 cardiomyocytes

(9400). c H9C2 cells show an intense staining localized on the cell

membrane with a punctuate diffuse pattern. d Negative control

(omission of primary antibody). e Magnification of c image.

f Detection of c-Mpl by flow cytometry in H9C2 cardiomyocytes.

g Indirect immunofluorescence staining for c-Mpl in isolated rat

ventricular cardiomyocytes as evaluated by confocal microscopy

(9600). Ventricular cardiomyocytes show an intense positive staining

localized on the cell membrane. At least three experiments were

performed for each experimental condition with similar results.

h Western blot analysis of c-Mpl expression in human heart. Lane 1Human platelet lysate (positive control); Lanes 2–4 Human heart

lysates


123

Finally, the expression of c-Mpl was evaluated in human

heart fragments by western blot (Fig. 1h).

Effect of thrombopoietin on papillary muscle

contractility

As shown in Fig. 2a, TPO had no direct effect on the

contractile force of rat papillary muscle. However, when

followed by EPI stimulation, TPO blunted the enhance-

ment of contractile force induced by EPI in a concentra-

tion-dependent manner (Fig. 2a). TPO, indeed, did not

change cardiac contractility at 25 and 50 pg/mL, but

induced a significant reduction of the effect of EPI at

100 pg/mL or higher concentrations (Fig. 2a). Nonetheless,

the effect of the highest concentrations of TPO tested (200

and 500 pg/mL) was comparable to that observed with

100 pg/mL TPO (Fig. 2a).

The ratio between the response of papillary muscles to

EPI at the end of each experiment and that recorded at the

beginning was close to 1 (1.02 ± 0.05), thus ruling-out

adrenergic receptor desensitization induced by repeated

challenges with EPI and alterations of cell viability.

Moreover, extensive washing of the papillary muscle with

Tyrode’s solution alone completely reversed the anti-

adrenergic effect of TPO (data not shown).

The specificity of TPO effect was assessed by blocking

the biological activity of TPO using a TPOR-Fc chimera [2,

34]. TPOR-Fc chimera did neither alter per se the con-

tractile force of papillary muscle nor influence its respon-

siveness to beta-adrenergic stimulation (Fig. 2b), but it

completely abrogated the effects exerted by TPO (100 and

200 pg/mL) on EPI-induced cardiac contractility (Fig. 2b).

Moreover, when we removed the endocardial endothe-

lium by Triton X-100 treatment, we observed a slight but

not statistically significant increase of the inotropic effect

induced by EPI in basal conditions, whereas the anti-

adrenergic effect exerted by TPO, although attenuated, was

still present (Fig. 2c).

Effect of thrombopoietin in isolated heart

As shown in Fig. 3, while TPO (200 pg/mL) in basal

conditions did not alter LVP, it blunted the enhancement of

contractile force induced by EPI (0.1 lmol/L) (Fig. 3).

TPOR-Fc chimera completely abrogated the effects exerted

by TPO on EPI-induced cardiac contractility in isolated

heart (Fig. 3).

Involvement of the PI3K-Akt1-NO synthase-guanylyl

cyclase pathway

Specific pharmacological inhibitors were used to evaluate

the role of the PI3K-Akt1-NO synthase-guanylyl cyclase

pathway in the alterations of cardiac response to EPI

induced by TPO (200 pg/mL). Pre-treatment of papillary

muscles with the PI3K inhibitor wortmannin significantly

Fig. 2 : Effect of TPO on papillary muscle contractility. a Effect of

increasing concentrations of TPO (25–500 pg/mL) on isolated rat

papillary muscle contractility in the absence (open columns) or

presence (dashed columns) of 3 lmol/L epinephrine (EPI). b Effect

of TPO (100–200 pg/mL) on isolated rat papillary muscle contrac-

tility in the absence (open columns) or presence (dashed columns) of

the TPO receptor-Fc chimera (1.25 lg/mL). Papillary muscles were

stimulated with epinephrine (EPI; 3 lmol/L). c Effect of removal of

endocardial endothelium by Triton X-100 on the anti-adrenergic

effect of TPO in isolated rat papillary muscle. Papillary muscles were

stimulated with epinephrine (EPI; 3 lmol/L). Tmax: peak mechanical

tension. In these groups of papillary muscles, baseline value of Tmax

was 136.7 ± 12.2 mg. Values are derived from five or more

experiments for each TPO concentration and expressed as percent

variations from baseline value


123

reduced the effect of TPO on EPI-induced increase of

contractility (Fig. 4a). ODQ, which blocks the activity of

guanilyl cyclase, exerted a similar effect, as well as the

inhibitor of NO synthase L-NAME (Fig. 4a). On the con-

trary, the biologically inactive enantiomer D-NAME did not

affect the action of TPO on EPI-induced contractility (not

shown). None of the drugs altered the responsiveness of

papillary muscles to EPI (not shown).

Direct phosphorylation of Akt1 in H9C2 cardiomyo-

cytes and papillary muscles challenged with TPO

(200 pg/mL), but not with Tyrode’s solution alone, was

also directly demonstrated by western blot (Fig. 4b, c). In

this experimental setting, insulin was used as positive

control to induce Akt1 phosphorylation in H9C2 cells

(Fig. 4b, c).

Patient clinical characteristics and TPO levels

Table 1 gives demographic and clinical data for patients

with septic shock and healthy subjects. Septic shock

patients did not differ regarding demographic characteris-

tics from healthy subjects. All patients required vasopres-

sors, and the intra-hospital mortality was 50% (3 out of 6).

Platelet counts were not different between the groups

(Table 1). Leukocyte counts were significantly higher in

patients with septic shock than in healthy subjects; in

contrast, absolute monocyte counts were not different

between the groups (Table 1). Serum TPO concentrations

were significantly higher in septic shock patients than in

healthy subjects (Table 1).

Fig. 3 Effect of TPO on isolated heart contractility. Effect of TPO

(200 pg/mL) on isolated rat heart contractility in the absence or

presence of epinephrine (EPI; 0.1 lmol/L), or of the TPO receptor-Fc

chimera (1.25 lg/mL). Rat hearts were perfused at constant flow

(7.8 ± 0.3 mL/min/g) through a peristaltic pump with oxygenated

(100% O2) Tyrode’s solution, and electrically paced at constant

frequency (280 beats/min). LVP left ventricular pressure. Baseline

values were: LVP = 98.7 ± 4.2 mmHg; left ventricular end-diastolic

pressure (LVEDP) = 5.5 ± 4.0 mmHg. Values are derived from five

independent experiments and expressed as percent variations from

baseline value

Fig. 4 a pharmacological modulation of the negative inotropic effect

of TPO in papillary muscle. Pharmacological inhibition of phospha-

tidylinositol 3-kinase (PI3K) with wortmannin (Wort; 100 nmol/L),

guanylyl cyclase with ODQ (10 lmol/L), or NG-nitro-L-arginine

methyl ester (L-NAME; 1 mmol/L) inhibits the anti-adrenergic effect

of TPO in isolated rat papillary muscle. Papillary muscles were

stimulated with epinephrine (EPI; 3 lmol/L). Tmax: peak mechanical

tension. Baseline value of Tmax for this group of papillary muscles:

156.7 ± 12.5 mg. Values are derived from three independent exper-

iments for each drug and expressed as percent variations from

baseline value. b TPO-induced Akt1 phosphorylation in papillary

muscle. Representative western blot and densitometric analysis

(n = 3) of Akt1 phosphorylation in isolated rat papillary muscle in

the absence (Ctrl) or presence of 200 pg/mL TPO. c TPO-induced

Akt1 phosphorylation in H9C2 cardiomyocytes. Representative

western blot and densitometric analysis (n = 4) of Akt1 phosphory-

lation in H9C2 cardiomyocytes in the absence (Ctrl) or presence of

500 pg/mL TPO. Insulin (100 nmol/L) was used as positive control to

induce Akt1 phosphorylation in H9C2 cells


123

Role of thrombopoietin in the negative inotropic effect

exerted by human septic shock (HSS) serum

HSS serum (10%) reduced the contractile force of papillary

muscles of about 50% (Fig. 5). This effect was observed

with all the six serum samples tested. On the contrary, no

change in contractile force was induced by serum (10%)

from healthy subjects (Fig. 5). TPOR-Fc chimera com-

pletely prevented the decrease in contractile force induced

by HSS serum alone, whereas no effect was observed when

TPOR-Fc chimera was pre-incubated with serum of healthy

subjects (Fig. 5).

We next evaluated whether TPO may cooperate with

TNF-a and IL-1b in depressing cardiac contractility. Both

TNF-a (10 ng/mL) and IL-1b (10 ng/mL) markedly

reduced papillary muscle contractility and the negative

inotropic effect of both cytokines was significantly ampli-

fied by the addition of TPO (200 pg/mL) (Fig. 6). TPOR-

Fc chimera did not modify the contractile responses

induced by TNF-a or IL-1b alone, but it completely pre-

vented the additional decrease in contractile force induced

by incubating TPO with these cytokines (Fig. 6).

Discussion

The present study shows that TPO negatively modulates

myocardial contractility in both rat papillary muscle and

isolated heart. This effect is mediated by the stimulation of

TPO receptor c-Mpl on cardiac myocytes and consequent

activation of the PI3K-Akt1-NO synthase-guanylyl cyclase

pathway. Moreover, we showed that TPO is instrumental in

the negative inotropic effect induced in vitro by HSS serum

and cooperates with both TNF-a and IL-1b, the main

cytokines responsible for the myocardial depressant effect

of HSS serum.

We demonstrated the presence of the TPO receptor

c-Mpl in the rat heart by RT-PCR and immunoblotting,

confirming what already reported by Baker and colleagues

[3]. In addition, we showed c-Mpl expression in human

myocardium by immunoblotting. Since endothelial cells

Fig. 5 Effect of TPOR-Fc chimera on the negative inotropic effect of

human septic shock (HSS) serum in papillary muscle. Effect of pre-

treatment with the TPOR-Fc chimera on the negative inotropic effect

exerted by serum from six patients with septic shock (HSS serum) or

from six healthy subjects (10%) in isolated rat papillary muscle. Tmax:

peak mechanical tension. Baseline value of Tmax for this group of

papillary muscles: 148.3 ± 14.0 mg. Values are expressed as percent

variations from baseline value

Fig. 6 Effect of TPO on the negative inotropic effect of TNF-a and

IL-1b in papillary muscle and inhibition by TPOR-Fc chimera. Effect

of TPO (100–200 pg/mL) on the negative inotropic effect exerted by

TNF-a (10 ng/mL; a) and IL-1b (10 ng/mL; b) in isolated rat

papillary muscle. Pre-treatment with the TPOR-Fc chimera com-

pletely prevented the decrease in contractile force induced by addition

of TPO to TNF-a and IL-1b. Tmax: peak mechanical tension. Baseline

value of Tmax for this group of papillary muscles: 166.1 ± 16.5 mg.

Values are derived from five independent experiments and expressed

as percent variations from baseline value


123

express c-Mpl [7], we studied the presence of c-Mpl also in

cultured cardiomyocytes, definitely showing that they

express c-Mpl on the cellular surface, as also confirmed by

confocal microscopy.

We then evaluated the effects of TPO on myocardial

contractility in vitro and found that TPO did not directly

modify the contractile force of isolated rat papillary mus-

cle, but blunted the enhancement of contractile force

induced by EPI in both papillary muscle and isolated heart

preparations. TPO indirect negative effect on myocardial

contractility in isolated papillary muscle started at 50 pg/mL

to increase at higher concentrations (100–500 pg/mL),

although it reached a plateau after 100 pg/mL. We previ-

ously observed similar TPO effects in other experimental

settings, where TPO exerts no direct action per se, but

rather amplifies the effects of other biological mediators.

For instance, TPO does not directly induce platelet acti-

vation, but primes platelet aggregation and monocyte–

platelet interaction in response to ADP and EPI [37], and

enhances the production of oxygen free radicals by poly-

morphonuclear leukocytes challenged with fMLP [6].

The specificity of TPO effect was assessed by inhibiting

the biological activity of TPO using a TPOR-Fc chimera,

which completely abrogated the effects exerted by TPO on

EPI-induced cardiac contractility, whereas did neither alter

per se the contractile force of papillary muscle or isolated

heart nor influence their responsiveness to beta-adrenergic

stimulation.

Adrenergic receptor desensitization was excluded since

the response to EPI recorded at the end of the experiments

was similar to that recorded at the beginning. Moreover, a

toxic effect of TPO on myocardial cell viability was

excluded since extensive washing of the papillary muscle

or the isolated heart with Tyrode’s solution alone com-

pletely reversed the anti-adrenergic effect of TPO.

It is known that anti-adrenergic effects on cardiac

muscle may be mediated by NO release from endothelial

cells [13, 16, 48]. When endocardial endothelium was

removed by Triton X-100 treatment, we observed a slight

increase of the inotropic effect induced by EPI, although

not statistically significant, which may be explained with

the fact that endothelial cells tonically produce and release

NO also in basal conditions. However, the anti-adrenergic

effect exerted by TPO was still present, although attenu-

ated, further suggesting that TPO may exert its negative

inotropic activity directly on cardiomyocytes, and that

endocardial endothelium plays only a minor role in medi-

ating the effects of TPO.

The occurrence of myocardial depression in septic shock

is a well-documented phenomenon, which is associated

with high mortality rate [27, 36, 42]. Interestingly, the

major pathogenic mechanisms of septic cardiac dysfunc-

tion include both alterations of adrenergic response and the

effects of humoral mediators [27, 28, 36, 42]. Myocardial

hyporesponsiveness to catecholamines (including

decreased chronotropy and inotropy) has been shown in

several endotoxic models of septic shock [1, 45], as well as

in HSS [4, 46]. This effect has been related to disruption of

b-adrenergic signal transduction in cardiomyocytes due to

both NO-dependent and NO-independent mechanisms [29],

analogously to what reported for ischemia/reperfusion

injury [19, 40, 43]. Our results suggest that also TPO may

influence the contractile response elicited by EPI stimula-

tion by affecting adrenergic signal transduction. Moreover,

the results obtained using specific pharmacological inhi-

bitors on isolated rat papillary muscles show that TPO

action is mediated by the activation of the PI3K-Akt1-NO

synthase-guanylyl cyclase pathway, which leads to the

production of NO as final mediator. None of the drugs used

altered the responsiveness of papillary muscles to EPI, thus

excluding any aspecific side effect of these drugs on

adrenergic stimulation. We have also shown that TPO

directly induces the phosphorylation of Akt1 in H9C2

cardiomyocytes and isolated papillary muscles.

Evidence of a circulating myocardial depressant sub-

stance in the serum of septic shock patients was first

demonstrated by Parrillo et al. [39]. Subsequent studies

indicated TNF-a and IL-1b as the pivotal mediators

inducing cardiac depression in sepsis [25] and other disease

conditions [12, 49]. On the basis of the recent reports of

elevated TPO levels in sepsis [11, 22, 47, 52], we

hypothesized that TPO may concur to depress myocardial

contractility during sepsis. We observed that pre-treatment

of serum samples with the TPOR-Fc chimera completely

prevented the decrease in contractile force induced by HSS

serum alone, whereas it had no effect on serum of healthy

subjects. In addition, we found that the negative inotropic

effect of both TNF-a and IL-1b was significantly enhanced

by the addition of TPO. Moreover, TPOR-Fc chimera

completely prevented the decrease in contractile force

induced by the addition of TPO to TNF-a or IL-1b,

whereas did not modify the contractile responses induced

by these cytokines alone. The results obtained strongly

suggest that high levels of circulating TPO in patients with

sepsis may favor the occurrence of myocardial depression

in cooperation with TNF-a and IL-1b.

Recent experimental studies reported results apparently

not consistent with ours [3, 32]. Li and colleagues [32]

showed that TPO, at pharmacological doses, has anti-

apoptotic activity in H9C2 and spontaneously beating

cardiomyocytes, and preserves cardiac functions, including

heart rate, fractional shortening, and cardiac output, eval-

uated by echocardiography, in a model of doxorubicin-

induced acute cardiotoxicity. In addition, Baker and

colleagues [3] demonstrated that TPO treatment prevents

the decline in ventricular function and reduces infarct size


123

following ischemia/reperfusion in the rat isolated heart. We

have no clear explanation for the discrepancy between

these results and ours, although the different experimental

models, together with the concentrations used, well above

the physiological range, may, at least partially, justify these

results. In our study, we evaluated indeed TPO cardiac

effects at doses analogous to those measured in human

pathology, in particular during septic shock [52]. More-

over, we studied the acute changes induced by TPO pre-

treatment on EPI-stimulated myocardial contractility in a

time-frame in which apoptosis could difficultly take place.

Finally, and most important, although it is well known that

TPO primes the activity of other mediators on important

biological effects in mature platelets and other cell types

[6, 37, 38, 50], previously reported results were obtained in

controlled experimental conditions, in which only the

effects of TPO alone and at high doses were considered,

while the combined effects of the administration of TPO

with other soluble mediators were not evaluated [3, 32]. On

the contrary, we studied the effects of TPO at physiologic

concentrations and in association with either an adrenergic

stimulus (EPI) or the main cytokines known to depress

myocardial activity in septic shock, i.e. TNF-a and IL-1b.

Therefore, our findings stress the importance of careful

evaluation of the cardiovascular effects of TPO in vivo,

especially in the clinical setting of diseases, as is septic

shock, whose pathogenesis is complex and involves the

activation of a cascade of soluble mediators. Interestingly,

large-scale clinical trials evaluating the effects of mono-

clonal antibodies against TNF-a in chronic heart failure

patients gave rather disappointing outcomes [5, 10], sug-

gesting that counterbalancing this cytokine alone may not

be sufficient [51]. The individuation of TPO as an addi-

tional molecular target may provide clue for the develop-

ment of new therapeutic interventions for the treatment of

patients with sepsis-associated cardiac dysfunction and

eventually chronic heart failure. Interestingly, several

recent studies also proposed new experimental therapeutic

approaches to various cardiac diseases based on the mod-

ulation of the inflammatory and pro-thrombotic states

which are often associated with their development and

progression [9, 17, 21, 33, 41].

In conclusion, we showed here that TPO negatively

modulates myocardial contractility by stimulating its

receptor c-Mpl on cardiomyocytes and the subsequent

production of NO. In addition, TPO mediates the cardio-

depressant activity exerted in vitro by serum of septic

shock patients, which is indeed completely abrogated by

the TPOR-Fc chimera. Finally, TPO cooperates with TNF-

a and IL-1b in depressing cardiac contractility. Taken

together, our results suggest that TPO may have a relevant

role in modulating cardiac inotropy in septic shock by

affecting the two major pathogenic mechanisms described:

(a) by influencing adrenergic receptor signal transduction

and (b) by cooperating with circulating mediators known to

reduce myocardial contractility, namely TNF-a and IL-1b.

The further elucidation of the pathogenic mechanisms of

myocardial dysfunction during septic shock may lead to the

development of additional options for therapy.

Acknowledgments This work was supported by Ministero

dell’Universita e della Ricerca Scientifica e Tecnologica (MURST)

ex-60% to GM and GA, and Progetto di Ricerca Sanitaria Finalizz-

ata—Regione Piemonte to GM, GA, and EL.

Conflict of interest statement The authors declare that they have

no conflict of interest.

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