J Mol Cell Cardiol 28, 881–892 (1996)

Vascular-derived Myocardial ContractileFactor: Positive Myocardial InotropicSubstance Released from Medial Layerof the Canine AortaKai Li1,2, Xiuling Qi1, Luc Andries3, Duncan Stewart4, Pierre Sirois2,Dirk Brutsaert3, and Jean L. Rouleau1

1Department of Medicine, Montreal Heart Institute, University of Montreal, Canada, 2Department ofPharmacology, University of Sherbrooke, 3Department of Physiology, University of Antwerpen,Belgium, and 4Department of Medicine, Royal Victoria Hospital, McGill University, Canada

(Received 25 July 1995, accepted in revised form 10 November 1995)

K. L, X. Q, L. A, D. S, P. S, D. B J. L. R. Vascular-derived MyocardialContractile Factor: Positive Myocardial Inotropic Substance Released from Medial Layer of the Canine Aorta.Journal of Molecular and Cellular Cardiology (1996) 28, 881–892. Interactions between the various cell types thatmake up the cardiovascular system are known to play an important role in maintaining homeostasis. One areaabout smooth muscle cells that has received little attention, despite the production of a wide variety of mediatorsby smooth muscle cells, is their effect on myocardial function. In this study, the myocardial contractile effects offour different types of dog aortic strips on rabbit papillary muscles were evaluated. Of these, medial vascularsmooth muscle strips most consistently (65% of the time) produced a “vascular-derived contractile factor” (VDCF),which caused a 15% increase in isometric twitch tension and a 24% increase in isotonic twitch shortening withno change in twitch configuration. Endovascular strips with or without intact endothelium and complete aorticrings had less consistent effects. Vascular-derived contractile factor was stable after freezing at−80°C, its activitywas not modified by a broad spectrum peptidase, but it was heat-labile. The angiotensin II blocker, losartan, didnot modify its effects. However, incubation with indomethacin did reduce, but did not eliminate, the contractileeffects of vascular strips. The addition of a1- and b-blockers did not further modify the effects of VDCF. Endocardialendothelial removal increased the effects of VDCF. No correlation existed between endothelin levels and thecontractile effects of vascular strips. It is concluded that VDCF is produced by the medial layer of large vesselsbut its exact cellular origin is uncertain. These findings expand the ever-increasing understanding of the inter-relationship between the structures that make up the cardiovascular system, and open the door to new studiesevaluating the inter-relationship of vessels and myocardium. 1996 Academic Press Limited

K W: Contractility; Vascular smooth muscle; Myocardium; Aorta; Inotropy.

1992; Li et al., 1994). Vascular endothelial cellsIntroductionhave been shown to modify the contractile char-acteristics of vascular and myocardial cells (Li andImportant interactions between the various cell

types that make up the cardiovascular system have Chen, 1987; Yanagisawa et al., 1988; Vanhouetteet al., 1991; Ramaciotti et al., 1992; Li et al.,been demonstrated (Furchgott and Zawadski, 1980;

Yanagisawa et al., 1988; Brutsaert, 1989; Li and 1993; Ramaciotti et al., 1993). Myocardial-derivedsubstances, such as atrial natriuretic peptide (ANF),Xu, 1989; Vanhoutte et al., 1991; Ramaciotti et al.,

Please address all correspondence to: Jean L. Rouleau, Department of Medicine, Montreal Heart Institute, 5000 Belanger Street,Montreal, Quebec, Canada H1T 1C8.

0022–2828/96/050881+12 $18.00/0 1996 Academic Press Limited881

K. Li et al.882

have been shown to have effects on vascular smooth (Brutsaert et al., 1988; Meulemans et al., 1988;Shah et al., 1989a; Shah et al., 1989b; Li et al.,muscle and endothelium (Sonnenberg, 1990; Kubo

et al., 1994), and vascular smooth muscle has been 1991; Wang et al., 1991; Li et al., 1993), themyocardial contractile effects of vascular medialshown to modify the secretory characteristics of

endothelium (Stewart et al., 1990). Whether cells preparations were evaluated in papillary muscleswith and without endocardial endothelium.of vascular tissue other than vascular endothelium

have myocardial contractile effects is unknown. In All four types of vascular preparations were foundto produce a substance that increased myocardialthis study, particular attention was paid to the

potential for the production of contractile effects by contractility, a substance termed “vascular-derivedcontractile factor” (VDCF) by the authors. As allthe medial layer of the aorta, which is mainly

comprised of smooth muscle cells. types of aortic preparations did not cause inotropiceffects consistently, it would appear that the pro-Vascular smooth muscle may exist in two states,

a predominantly contractile state and a pre- duction of VDCF is controlled by some externalfactors, one of which may be related to the tech-dominantly secretory state (Casscells, 1991; Kasuya

et al., 1993; Stiemer et al., 1993). In its secretory nique of harvesting of the aortic preparations. Thefact that the medial smooth muscle layer pre-state, vascular smooth muscle produces a wide

range of substances (Takayasu-Okishio et al., 1990; parations produced VDCF most consistently, wouldsuggest that VDCF is produced by vascular smoothScott-Burden et al., 1991; Kasuya et al., 1993;

Stiemer et al., 1993; Schini et al., 1994; Tomlinson muscle cells. However, a vascular smooth musclecell origin for VDCF cannot be made with certainty.et al., 1994). Some of these, such as nitric oxide or

the products of the cyclooxygenase pathway, havepotential contractile effects (Okishio et al., 1990;Abete et al., 1992; Schror and Hohlfeld, 1992; Materials and MethodsSchini et al., 1994; Tomlinson et al., 1994). In thisstudy, the in vitro myocardial contractile response Isolated papillary muscle preparationsto vascular tissues freshly harvested from dogswere evaluated. These freshly harvested vascular New Zealand white rabbits weighing 2.5±0.5 kg

were anesthetized with 25 mg/kg of nembutal intra-preparations were made up of four types of canineaortic preparations. The first was that of whole venously. The heart was excised, the right ventricle

opened, and a papillary muscle was removed andvascular rings. This was done to evaluate the myo-cardial contractile response to all cell types that mounted in a bath filled with Krebs–Henseleit so-

lution. Eight right ventricular muscles from 10make up the aorta. The second consisted of en-dovascular preparations which included vascular mongrel dogs weighing 25±2 kg were also chosen

for mechanical studies. Dogs were anaesthetizedendothelium, its sublayer of connective tissue, andvarying amounts of vascular smooth muscle. This with 25 mg/kg of nembutal intravenously, their

chest opened, their heart removed, and a rightpreparation was used to maximize the myocardialcontractile response to the internal layer of the ventricular papillary muscle or trabeculae removed

and mounted in a bath. Only muscles longer thanaorta. The third preparation was identical to thesecond, except that the endothelial layer was re- 4 mm and with a cross-section <0.8 mm2 were

chosen for the study.moved. This preparation was used in order to betterappreciate the myocardial contractile response to The base of the muscle was held by a stainless

steel clamp with the other end tied to a lever withvascular endothelium, as any loss of contractileeffects in this preparation as compared to the former an electromagnetic feedback system identical to

that of Brutsaert et al. (1973) to allow control ofwould have supported an important endothelialrole. The fourth preparation consisted of the vas- force, length and velocity. The muscles were bathed

in Krebs–Henseleit solution containing (m): so-cular medial layer which is largely composed ofvascular smooth muscle. This preparation has the dium chloride (NaCl), 118; sodium bicarbonate

(NaHCO3), 29.9; potassium chloride (KCl), 3.5;advantage of maximizing the myocardial contractileresponse to vascular smooth muscle. The myo- magnesium sulphate (MgSO4), 1.2; calcium chloride

(CaCl2), 1.25; potassium phosphate (KH2PO4), 1.2;cardial contractile effects in rabbit and dog papillarymuscles after the addition of these four aortic pre- dextrose, 4.5 ph. This solution was bubbled with a

gas mixture of 95% O2/5% CO2 at a temperatureparations were evaluated. Due to recent reportsindicating that endocardial endothelium modifies of 29°C and pH of 7.4.

The muscles were stimulated 10% above thresh-myocardial contractility and also modifies the myo-cardial contractile effects of circulating substances old with a Grass S-88 stimulator through platinum

Myocardial Effects of Vascular-derived Contractile Factor 883

field electrodes at 12 stimuli/min for rabbit musclesand at 6 stimuli/min for dog muscles. In all ex-periments, the preload on the muscle was adjustedso that initial muscle length was at Lmax (lengthat which maximal developed tension occurred).

Aorta out and wash

5 min

Aorta in

Muscles were permitted to stabilize at Lmax for 2 hFigure 1 Representative curve showing the increase inand isometric and isotonic contractions were re-peak twitch shortening of a rabbit papillary muscle in-corded on a Gould 2400 S recorder. After adjustingduced by the addition of an aortic strip to the bath,

the elastic damping of the force–length–velocity followed by a decrease in shortening as a result of removallever feedback system to compensate for electro- of the vascular smooth muscle strip and washing of themechanical transients, the maximum velocity of bath with Krebs–Henseleit solution.unloaded muscle shortening (Vmax) was obtained byabruptly decreasing the load on the muscle at

surface up, onto a wax-based steel basin. A cross-the time of activation (zero load clamp). Theseincision just below and parallel to the endothelialisometric, isotonic and unloaded contractions werelayer starting at the proximal end of the aortic stripused as baseline values. The output of the amplifierwas performed and extended throughout the aorticof the Gould 2400 S recorder was connected to anstrip. Great care was taken to avoid touching orelectronic differentiator and both of these were feddamaging the endothelial layer. This endothelialinto an A/D converter (Data Translation, DT 2821-and subendothelial preparation was termed “en-F-801; Marlborough MA, USA), which in turndovascular strip with intact endothelium”. The thirdwas connected to a COMPAQ Deslepco 286aortic preparation was similar to the second, how-microcomputer. Analysis of force-length char-ever, once the vascular endothelial preparation wasacteristics was performed by custom-made softwareobtained, endothelial cells were removed by mech-running under MS-DOS. The muscle cross-sectionanically rubbing the preparation with a surgicalwas measured by assuming the muscles to be ofblade. This yielded a preparation with only thecylindrical shape and dividing muscle weight bysubendothelial layer and was termed “endovascularlength.strip without endothelium”. The fourth aortic pre-Two types of isolated rabbit papillary muscleparation was comprised of medial vascular smoothpreparations were used; (1) with intact endocardialmuscle strips. These were obtained by dissectingendothelium, and (2) after endocardial endotheliumout the endothelial and subendothelial layers asremoval. Endocardial endothelium was removed bydescribed above, and dissecting away the adventitiarapid immersion of papillary muscles into a 1%on the outer side of the aortic strip. This preparationTriton X-100 solution. Muscles were then washedwas termed “medial vascular smooth muscle strip”.abundantly in oxygenated Krebs–Henseleit solution

and permitted to restabilize for 2 h.

Morphological studies

Preparation of aortic strips Scanning electron microscopy (SEM), confocal scan-ning light microscopy (CSLM) and light microscopy

Mongrel dogs weighing between 20 and 30 kg (LM) were used to detect morphological changes inwere anaesthetized with thiopental (25 mg/kg) and vascular strips prior to and after surgical bladeventilated mechanically. The chest was opened via rubbing. The viability of the endothelial cells wasa paramedian incision, and an 8-cm ring of the verified by CSLM using, prior to fixation, propidiumascending aorta was removed and immediately iodide to stain nuclei of dead cells. After fixation,washed 5–10 times in oxygenated Krebs–Henseleit F-actin in vascular endothelial and vascular smoothsolution in order to eliminate potential modulating muscle cells was stained by Bodipy-phallacidin.substances from serum that may have adhered to These methods have been described in detail else-the aortic strips. The aorta was then placed in a where (Li et al., 1993a; Li et al., 1993b).bath with oxygenated Krebs–Henseleit solution andwas opened via a longitudinal incision.

Four types of aortic strip preparations were used Protocol A: Effect of various aortic strip preparations onmyocardial contraction(Fig. 1). The first comprised of intact aortic rings.

The second was a preparation that included theAs pilot studies indicated that endocardial endo-endothelial and subendothelial layers. In order to

do this, the aortic strip was pinned, endothelial thelial removal increased the myocardial contractile

K. Li et al.884

effects of VDCF, rabbit papillary muscles without Chemicals Co., St Louis MO, USA). Prior to testingthe effects of this peptidase on the myocardial con-endocardial endothelium were used for this portion

of the study. Once papillary muscles had been stable tractile effects of active solution, the potential toxicmyocardial effects of this peptidase were evaluatedfor at least 1 h, isometric, isotonic and unloaded

(Vmax) contractions were recorded. Intact aortic on five rabbit papillary muscles with or withoutendocardial endothelium. After measuring baselinerings (n=8), endovascular strips with intact endo-

thelium (n=112), endovascular strips without isometric, isotonic and unloaded muscle con-traction, peptidase was added to the bath in in-endothelium (n=14), or medial vascular smooth

muscle strips (n=110) were added to the papillary creasing doses, 0.005, 0.05 and 0.5 units, andcontractions were measured at each dose. As pep-muscle bath and isometric, isotonic and unloaded

contractions were recorded 60 min later. The aortic tidase 0.5 units did not significantly alter contractilecharacteristics, this dose was used to assess thestrips were then removed, the liquid from the bath

collected and frozen at −80°C, and the muscles effects of peptidase on the stability of the myocardialcontractile effects of active solution. Peptidase 0.5washed profusely. Muscles were permitted to re-

stabilize for 1 h and repeat (re-baseline) isometric, units was added to 20 ml of active solution whichhad been in the bath for 1 h and had caused aisotonic and unloaded contractions were recorded.

Muscles were classified into responders and non- significant increase in tension. One hour later, re-peat values were recorded with the peptidase andresponders according to whether tension generation

increased >5% with the introduction of the aortic active VDCF solution together. This was followedby profuse washing and re-baseline values werestrips.

In six rabbit papillary muscle preparations in recorded, later.which the addition of an aortic strip led to anincrease in twitch isometric tension generation, asecond control papillary muscle preparation with- Protocol C: Influence of endocardial endothelium on the

effect of VDCFout the addition of an aortic strip was used as acontrol. After 1 h of incubation and once maximal

In order to assess whether the effects of activemyocardial contractile effect was attained, the aor-tic strip was removed and the active solution trans- solution were modified by endocardial endothelium,

eight rabbit papillary muscles with intact en-ferred to the control bath. The other papillarymuscle preparation was then bathed with the docardial endothelium and eight rabbit papillary

muscles without endocardial endothelium were firstKrebs–Henseleit solution from the control bath.They were washed profusely and then repeat values stabilized for 4 h. Baseline, isometric, isotonic and

unloaded contractions were recorded. Two 20-mlfor both papillary muscle groups were recorded 1 hlater. aliquots of the most active solutions were chosen,

mixed together, and re-separated into 20-ml al-iquots. These were added to each of the bathsinstead of the control solution. One hour later,Protocol B: Stability studies of VDCFrepeat contractions were recorded. All muscles werethen washed and re-baseline values were obtainedThe stability of liquid from the bath of muscles that

responded to aortic strip insertion (active solution) 1 h later. As endocardial endothelial removal in-creased the effects of VDCF, except for studies onwas evaluated in three ways. First, liquid frozen at

−80°C was thawed and added to the bath of five dog papillary muscles, all other studies were doneon papillary muscles without endocardial endo-rabbit papillary muscles. Isometric, isotonic and

unloaded contractions were recorded 1 hour later thelium.and compared to baseline values. Second, activesolution from two aortic strip preparations wascombined and reseparated into two 20-ml aliquots. Protocol D: Effect of VDCF on dog myocardium and the

effect of losartanOne of the two aliquots was heated at 100°C for30 min and added to a bath with a rabbit papillary

In order to assess whether active solution alsomuscle. The other aliquot served as a control andwas added to a second bath with a rabbit papillary modified the contractile characteristics of dog myo-

cardium, papillary muscles with intact endocardialmuscle. This was repeated on three separate oc-casions. The third evaluation of the stability of endothelium from five dogs were used. Once har-

vested and mounted in an isolated bath, musclesactive solution included exposure to a peptidasefrom porcine intestinal mucosa (# P7500, Sigma were permitted to stabilize for 3 h prior to obtaining

Myocardial Effects of Vascular-derived Contractile Factor 885

isometric, isotonic and unloaded contractions. The oxygenated Krebs–Henseleit solution with no drugadded. After 30 min incubation, the aortic stripscontrol solution was replaced with 20 ml of thawed

active solution for 1 h and then 5 l of losartan were added to the bath with papillary musclesbathed in a solution similar to that in which thewas added. Subsequently, papillary muscles were

washed profusely after 1 h infusion with the an- aortic strip was incubated. Isometric and isotoniccontractions were recorded prior to and after 30 mingiotensin II type 1 (AT-1) receptor blocker, losartan,

permitted to restabilize for 1 h and re-baseline val- of incubation of aortic strips with the papillarymuscles. The addition of ASA changed the pH ofues recorded.the Krebs–Henseleit solution from 7.48 to 7.38.The addition of ASA did not result in a modificationin contractile characteristics.Protocol E: Effect of indomethacin, a1-and b-adrenergic

blockade and acetylsalicylic acid (ASA) on the effects ofVDCF

Protocol F: Stimulation of release of phagocytevasoactive substancesIn order to assess whether active solution acted via

prostaglandins or via a1-adrenergic or b-adrenergicThe variability in obtaining a positive inotropicreceptors, 28 vascular smooth muscle strips were

divided into two parts. One part of the strips was response could result from the variability of therelease of vasoactive substances from phagocytesincubated in an oxygenated Krebs–Henseleit so-

lution with a mixture of indomethacin 0.1 l, in the vascular strips. To investigate this hypothesis,f-Met-Leu-Phe (100 n) (Marceau et al., 1990) wasprazosin 0.1 l and propanolol 0.1 l for 30 min

prior to being added to a bath with the same mixture added to the bath of six rabbit papillary muscles1 h after addition of canine medial vascular smoothbathing a papillary muscle. The other part of the

strips was left in oxygenated Krebs–Henseleit so- muscle strips to the bath. Isotonic, isometric andunloaded contractions were recorded prior to add-lution without the mixture of blockers and then

added to a bath without the mixture of blockers. ing the vascular strip (baseline), 1 h after addingthe vascular strip and again 1 h after having addedIsometric, isotonic and unloaded muscle con-

tractions were recorded prior to the addition of the f-Met-Leu-Phe (100 n).vascular smooth muscle strip, after their addition,and after removing the strip and washing themuscle profusely. Muscles in the group treated with Protocol G: Endothelin measurements in solution

containing VDCFblockers were rinsed with a solution containingthese blockers.

Endothelin (ET) levels were determined by a mo-In order to verify if indomethacin was the activeblocker, a second group of 20 aortic strips was dification of a previously described radio-

immunoassay in the solution of aortic stripseparated into two parts. One part was incubatedin oxygenated Krebs–Henseleit solution with indo- preparations with varying degrees of contractile

activity (Cernacek and Stewart, 1989). Samplesmethacin 0.1 l and the other part was incubatedin oxygenated buffer with the vehicle for indo- were extracted with SepPack C18 cartridges

(Waters, Mississauga, Ontario, Canada) activatedmethacin (alcohol). After 30 min incubation, theaortic strips were added to the bath with papillary with methanol 8 mol/l urea, and water, and eluted

with methanol (recovery 75±3%). Samples andmuscles bathed in a solution similar to that inwhich the aortic strip was incubated. Isometric and standards (ET-1, Peninsula Laboratories, Belmont

CA, USA) were reconstituted in assay buffer andisotonic contractions were recorded prior to andafter 30 min of incubation of aortic strips with the incubated for 24 h with anti-ET-1 antibody (Pen-

insula Laboratories) at 4°C. The addition of 4000 ct/papillary muscles. Neither alcohol, nor indo-methacin alone, had any effect on myocardial con- min of [125I]ET-1 was followed by a second 24-h

incubation. Bound radioactivity was separated fromtractility.As indomethacin can have effects on calcium free radioactivity by the second antibody method

and evaluated after logit/log transformation.mobilization (Northover, 1972; Northover, 1973;Jeremy et al., 1990), a third group of 14 aortic Immunoreactive ET-1 (irET-1) is presented after

correction for recovery.strips was separated into two parts. One part wasincubated in oxygenated Krebs–Henseleit solution The antibody exhibited a cross-reactivity of 10%

with human “big” ET-1 and 5% with ET-3, butwith ASA 1 m (Rahmani et al., 1993) instead ofindomethacin, and the other part was incubated in no cross-reactivity with unrelated peptides. The

K. Li et al.886

standard curve was very stable with a midpoint the type of preparation being evaluated (Table 1).The aortic preparations deemed to have a negative(IC50) of 7.23±0.58 pg/tube. The limit of detection,

defined as the least amount of irET-1 distinguishable myocardial contractile response, nevertheless, res-ulted in an average increase in peak twitch tensionfrom zero at a 95% confidence level, was 0.12 pg/

tube. The intra- and inter-assay coefficients of vari- generation of 3.3%. Medial smooth muscle stripshad a greater incidence of a positive myocardialation were 9% and 12%, respectively. Serial di-

lutions of the fluid extract inhibited binding of contractile response (65%) than did the other pre-parations (Table 1). This increase in contractileradioligand in parallel with the standard curve,

and HPLC analysis of fluid extract demonstrated a indices started shortly after the addition of the aorticstrips and peaked within 1 h (Fig. 1). The effectdominant peak of irET-1 coeluting with synthetic

ET-1. could be washed out and was transferable fromone bath to another. Although contractile indicesincreased in response to this VDCF, the pattern ofthe twitch did not change significantly (Table 2,Fig. 2).

Results

Protocol B: Stability studies of VDCFMorphology of various aortic strip preparations

Freezing at −80°C did not alter the myocardialIntact aortic rings had viable endothelial andcontractile effects of active solution with VDCFsmooth muscle cells, as was shown by the presence(solution that caused a[5% increase in developedof actin staining and the absence of nuclei labeledtension). Prior to freezing, fresh solution caused anby propidium iodide. Endovascular strips with intactincrease in tension of 0.51±0.08 g/mm2 and, afterendothelium showed regions of damaged endo-being frozen then thawed, the increase in tensionthelial cells with nuclei stained by propidium iodide,was similar, i.e. 0.46±0.11 g/mm2. Heating atand regions with intact endothelial cells. The in-100°C for 10 min did, however, completely elim-timal surface of endovascular strips without endo-inate the myocardial contractile effects of activethelium was devoid of viable endothelial cells.solution (tension from 2.16±0.17 toExcept for some scattered damaged endothelial cells2.02±0.29 g/mm2 in heated solution) as comparedwith labeled nuclei, large areas of the intimal surfacewith control active solution (tension fromwere denuded and had a dark appearance which1.92±0.21 to 2.27±0.34 g/mm2). Finally, the ad-contrasted heavily with the actin-rich sub-endo-dition of peptidase to the bath did not modify thethelial resident smooth muscle cells. Some of thecontractile effects of vascular smooth muscle stripslatter cells were damaged. Medial smooth muscle(Fig. 3).cells in this protocol were intact, apart from some

damaged cells along the cut edges. Similarly, dam-aged smooth muscle cells were also found in medial

Protocol C: Influence of endocardial endothelium on thevascular smooth muscle strips along the cleavedeffect of VDCFand cut edges. Elsewhere, smooth muscle cells were

viable.The contractile effects of solution with VDCF weregreater in papillary muscles without endocardialendothelium as compared with those with en-docardial endothelium (Table 2, Fig. 2). However,in both situations, contractility increased withoutProtocol A: Effect of various aortic strip preparations on

myocardial contraction causing significant changes in twitch configuration.

For the purposes of this paper, a positive myocardialcontractile response to an aortic strip preparation Protocol D: Effect on dog myocardium and the effect of

losartanis defined as an increase in total peak twitch gen-eration of >5% of baseline. All responsesΖ5% are

The contractile effects of solution with VDCF hadconsidered to be negative myocardial contractileresponses. The addition of various aortic strip pre- similar effects on dog papillary muscles with intact

endocardial endothelium as it did in rabbit papillaryparations resulted in a positive myocardial con-tractile response 39–65% of the time according to muscles with intact endocardial endothelium. Total

Myocardial Effects of Vascular-derived Contractile Factor 887

Table 1 Positive myocardial inotropic effect induced by various aortic strip preparations

Positive response Basal TT Peak TT Change(%) (n for positive (g/mm2) (g/mm2) in TT (%)response: negative

response)

Aortic ring 50 (4:4) 2.11±0.20 2.38±0.21 12.8Endovascular strip 39 (44:68) 2.36±0.06 2.58±0.07 9.2with endotheliumEndovascular strip 43 (6:8) 2.31±0.21 2.57±0.24 11.1withoutendotheliumMedial smooth 65 (71:39) 2.38±0.06 2.73±0.09 14.7muscle strip

Change in TT (%) represents the change of all muscles studied (%) whether they had a positive ornegative response. TT, total peak isometric twitch tension generated, values are mean±.. A positiveresponse is defined as an increase of [5% in developed tension with the introduction of the aortic strip.

Table 2 Effect of active solution with vascular-derived contractile factor (VDCF) on myocardial contractionof rabbit papillary muscles with and without endocardial endothelium

Endocardial endothelium intact Endocardial endothelium removed(n=8) (n=8)

Baseline VDCF D Baseline VDCF D

TT (g/mm2) 2.22±0.19 2.72±0.28∗ 0.50±0.18 1.74±0.12 2.77±0.33∗ 1.03±0.30†dT/dt (g/mm2/s) 8.44±1.06 11.51±1.74∗ 3.07±1.41 6.88±0.65 13.56±2.65∗ 6.68±2.57†TTPT (ms) 258±14 251±16 −7±9 238±9 242±11 4±5RT1

2 (ms) 173±11 164±14 −9±12 159±8 156±13 −4±6Vmax (Lmax/s) 1.06±0.06 1.28±0.12 0.22±0.10 1.05±0.09 1.51±0.25∗ 0.46±0.18Shortening 9.4±1.7 12.4±1.9∗ 3.0±1.1 9.1±1.2 15.9±2.0∗ 6.8±2.0†(% Lmax)

Active solution is solution from aortic strip preparation causing an increase in peak isometric twitch tension of >5%. D,difference between solution with VDCF and baseline; TT, total peak isometric twitch tension; dT/dt, maximal rate of twitchtension development; TTPT, time from initiation of twitch peak twitch tension development; RT1/2, time from onset oftwitch to half isometric relaxation time; Vmax, maximal rate of shortening of an unloaded contraction; Lmax, length at whichthe muscle developed maximal twitch tension. ∗P<0.05 v baseline, †P<0.05 v intact endocardial endothelium.

600

3

Time (ms)

Ten

sion

(g/

mm

2 )

500

2

1

100 200 300 4000

+VDCF

–EE

600

3

Time (ms)500

2

1

100 200 300 4000

+VDCF

+EE

Figure 2 Representative curves of the effect of the same active vascular-derived contractile factor (VDCF) solution ontwitch configuration in rabbit papillary muscles with endocardial endothelium (+EE) and without endocardialendothelium (−EE). The effects of VDCF were slightly greater in muscles without endocardial endothelium.

K. Li et al.888

of blockers reduced, but did not completely prevent,the increase in tension (from 17.3% to 8.2%,P<0.05) caused by the addition of aortic smoothmuscle strips. Indomethacin alone or ASA alonehad, with respect to the response to the addition ofaortic smooth muscle strips, an effect similar to themixture suggesting that the only active blockers inthe mixture were those blocking the cyclooxygenasepathway.

4

0Baseline

Ten

sion

(g/

mm

2 )

+Activesolution

Wash

3

1

**

2

Peptidase

Protocol F: Stimulation of release of phagocyteFigure 3 Effects of peptidase on the myocardial con-tractile effects induced by vascular-derived contractile vasoactive substancesfactor (VDCF). The addition of 0.5 l peptidase did notmodify the myocardial contractile response of rabbit pap- The addition of f-Met-Leu-Phe to aortic medialillary muscles to active solution with VDCF (>5% increase

smooth muscle strips incubated with rabbit pap-in peak isometric twitch tension). Where ∗P<0.05 v.illary muscles had no effect on the contractile effectsbaseline.of these aortic preparations whether these pre-parations had induced a positive contractile re-sponse or not. The addition of f-Met-Leu-Phe didnot alter tension development (2.40±0.22 g/mm2

prior to addition and 2.35±0.20 g/mm2 after theaddition of f-Met-Leu-Phe).

Protocol G: Endothelin concentrations in solutioncontaining VDCF

Endothelin levels in active fluid were found to be

200

0Baseline

Ten

sion

(g/

mm

2 )

+Activesolution

Wash

150

50

**

100

+Losartan

1000 times lower than levels found to increaseFigure 4 Effects of losartan on the myocardial con- myocardial contractile indices (Fig. 6) (Li et al.,tractile effects induced by vascular-derived contractile 1991). Also, no correlation between change in peakfactor (VDCF). The addition of 5 l of losartan did not twitch tension and endothelin levels could be found.modify the myocardial contractile response of dog pap-illary muscles to active solution with VDCF (>5% increasein peak isometric twitch tension).

Discussion

This study demonstrates that vascular smoothtension increased from 5.3±1.4 to 6.3±1.7 g/muscle strips from freshly harvested dog aortasmm2 (P<0.01), dT/dt increased from 17±5 toproduce a positive inotropic substance or substances24±7 g/mm2/s (P<0.01), and Vmax increased fromthat have been termed “vascular-derived contractile1.5±0.11 to 1.7±0.08 Lmax/s (P<0.05), while timefactor” (VDCF). For reasons that could not be iden-to peak tension did not change. The addition oftified, fresh intact aortic preparations produced sig-losartan did not modify the contractile effects ofnificant inotropic effects only 39–65% of the timeactive solution containing VDCF (Fig. 4). As independing on the aortic preparation being used. Therabbit papillary muscles, after washing with Krebs-most consistent effects occurred when the medial orHenseleit solution, contractile characteristics re-vascular smooth muscle layer of the aorta wasturned to baseline values.being used, suggesting that VDCF may be producedby vascular smooth muscle cells. Nevertheless, onceproduced, it was stable and transferable. The pos-itive inotropic effects of VDCF were partially, butProtocol E: Effect of indomethacin, a1- and b-adrenergic

blockade not completely, blocked by indomethacin and ASA,suggesting that there might be two substances, one

The addition of the mixture of blockers slightly which is produced via the cyclooxygenase pathwayand one that is not. As a1 and b-adrenergic blockadelowered baseline total tension (Fig. 5). The mixture

Myocardial Effects of Vascular-derived Contractile Factor 889

3

0Baseline

Ten

sion

(g/

mm

2 )

+VSM Wash

2

1

(a)

Baseline +VSM Wash

(b)

*

†*

*

†*

Baseline +VSM

(c)

†*

*

Figure 5 (a) The combination of indomethacin, prazosin and propranolol reduced but did not abolish the myocardialcontractile response to medial smooth muscle (VSM) strips. Solid bars, control; open bars, three blockers. The effects of(b) indomethacin alone and (c) acetylsalicylic acid (ASA) alone (solid bars, control; open bars, 1 l ASA) were similarto the combined effects of all three blockers. +VSM (solid bars, control; open bars, 1 l indomethacin), after additionof vascular smooth muscle strip; ∗P<0.05 for +VSM v baseline; †P<0.05 for change of control v blockers with theaddition of VSM.

one animal to another, whether the gene or genesare present or not, and whether an animal ishomozygote or heterozygote for the gene or genes.As special precautions to protect against ischaemiaor damage to the aortic preparations appeared tohave little or no effect on results, it would appearthat cellular damage was not the major mechanismdetermining whether VDCF was produced in sig-nificant quantities.

The most likely cellular origins of VDCF is vas-cular smooth muscle. Vascular smooth muscle can

3

∆Tension (g/mm2)

irE

ndo

thel

in–1

(pM

)

0.7

2

0

–0.1 0.1 0.3 0.5–1

1

exist in a secretory as well as a contractile state,and is known to have the capacity of producingFigure 6 The concentration of irEndothelin-1 in so-

lutions containing vascular-derived contractile factor numerous substances (Takayasu-Okishio et al.,(VDCF). No correlation was found between the con- 1990; Casscells, 1991; Scott-Burden et al., 1991;centration of endothelin and the increase in peak twitch Kasuya et al., 1993; Stiemer et al., 1993; Schini ettension development.

al., 1994; Tomlinson et al., 1994) that could modifymyocardial contractility. Some of these substances

and AT-1 receptor blockade did not modify its con- are reasonably unstable and can be ruled out astractile effects, it is unlikely that VDCF exerts its making up a significant portion of VDCF. Otherpositive inotropic effects via these receptors. Also, substances are more stable and could account, atas a wide spectrum peptidase did not modify its least partially, for the effects of VDCF.effects, it is unlikely that VDCF is a peptide. Also, It is clear from the results of this study thatVDCF does not appear to be endothelin and does inotropic substances other than those produced bynot originate from endothelial cells. Thus, this study the endothelium have significant contractile effects.indicates that vascular substances other than those Vascular endothelium removal had no effect on thewhich are endothelial-derived can modify myo- production of VDCF. Also, endothelin levels werecardial contractility. present in concentrations <1000 times lower than

None of the vascular preparations studied con- those necessary to have inotropic effects (Li et al.,sistently produced VDCF in significantly contractile 1991) and, when present, no correlation betweenquantities. The reasons for this inconsistency are their concentration and the positive inotropic effectsuncertain and may be related to a need for in of vascular strips could be identified. Thus, VDCFvivo preconditioning of the cells producing these does not appear to originate from vascular endo-substances by neurohumoral, biochemical or phys- thelial cells.ical factors. Another possibility is that VDCF pro- In addition to vascular smooth muscle cells, the

vascular medial layer contains other componentsduction is genetically determined and varies from

K. Li et al.890

such as phagocytes and fibroblasts. Phagocytes in consistent with VDCF having its effects via in-creasing intracellular calcium. Endocardial endo-vascular tissue have been shown to release a vaso-

active substance which has potential myocardial thelial removal is known to reduce myofibrillaraffinity to calcium, thereby sensitizing myocardialcontractile effects (Marceau et al., 1990). However,

the authors’ studies with f-Met-Leu-Phe, which cells to changes in intracellular calcium con-centrations (Wang and Morgan, 1992). Thus, ifstimulates the release of cyclo-oxygenase products

such as thromboxane B2 from phagocytes, had no VDCF acts by increasing intracellular calcium, onewould have expected the removal of endocardialeffect suggesting that VDCF does not originate from

phagocytes. Another possible source for VDCF, endothelium to amplify the contractile effects ofVDCF, which it did in this study.which cannot be ruled out, is the connective tissue

matrix of the medial layer of the aorta. Numerous That indomethacin and ASA reduce, but do noteliminate, the inotropic effects of vascular strips,substances are known to be present in this matrix,

some of which are produced by fibroblasts and some suggests that VDCF could be composed of at leasttwo substances, one that is produced via the cyclo-of which are produced by other cell types (Jukkola

et al., 1991; Dell’Orbo et al., 1992; Stiemer et al., oxygenase pathway and another that is not. An-other possibility is that, despite using very large1993). However, at this time, no known substances

that could be VDCF are known to be present in doses of indomethacin and ASA, inhibition of thecyclo-oxygenase pathway was incomplete and that,the connective tissue matrix or be produced by

fibroblasts. Rather, these substances appear to have in fact, all constituents of VDCF are produced viathe cyclo-oxygenase system. A third possibility islittle or no contractile potential.

The exact chemical characteristics of VDCF were that the partial blocking effects of indomethacinare the results of its inhibitory effect on membranenot determined. However, it does not appear to be

a peptide, as a broad spectrum peptidase did not calcium mobilization (Northover, 1972; Northover,1973; Jeremy et al., 1990), rather than due to anprevent its contractile effects. Nevertheless, that

VDCF is a peptide cannot be completely ruled out, effect on the cyclo-oxygenase system. However, thatASA had the same effects as indomethacin suggestsas the lack of effect of the pig intestinal peptidase

used in this study may have been the result of some that this is not the case and that at least part ofVDCF is produced via the cyclooxygenase system.active fragments of vascular-derived contractile fac-

tors. The authors’ studies also demonstrate that In summary, this study demonstrates that themedial layer of the aorta produces a substance orVDCF is stable, can be frozen then thawed, and is

heat-labile. These stability characteristics rule out substances that affect myocardial contractility. Thisobservation suggests that more interaction thana number of unstable substances, such as nitric

oxide or unstable prostaglandins, as making up appreciated previously occurs between cells of vari-ous cardiovascular structures. What role, if any,significant portions of VDCF. The authors’ studies

with various antagonists suggest that the con- these substances have in maintaining cardio-vascular homeostasis remains to be determined,tractile effects of VDCF do not act through AT-1,

a1- or b-adrenergic receptors, as the use of blockers but the possibility that it may play a role, haemo-dynamic or cellular, warrants further investigation.did not appear to significantly alter the contractile

effects of vascular strips.Vascular strips caused contractility to increase

without significant alterations to twitch con- Acknowledgementsfiguration. These changes are in keeping with anintervention that increases intracellular calcium

The authors are indebted to H. Gosselin for per-without significantly altering activity of the phos-

forming the pilot studies and for his expert technicalphoinositol or adenylyl cyclase pathways (Li et al.,

assistance.1991; Wang et al., 1991; Li et al., 1993). This lackof effect on twitch configuration is thus consistentwith the present findings of a lack of effect ofAT-1, a1- and b-adrenergic receptor blockade, in- Referencesterventions that should have modified twitch con-

A P, F N, L D, C P, L P,figuration had VDCF had its actions via one of theseS S, B R, R F, 1992. Age-related effectsreceptors (Endoh and Blinks, 1988; Endoh et al.,of platelet activating factor (PAF) in the isolated per-

1991; Li and Rouleau, 1991; Li et al., 1991). fused rat heart. J Mol Cell Cardiol 24: 1399–1404.Finally, the greater effect of VDCF on papillary B DL, 1989. The endocardium. Ann Rev Physiol

51: 263–273.muscles without endocardial endothelium is also

Myocardial Effects of Vascular-derived Contractile Factor 891

B DL, C VA, G MA, 1973. Effects papillary muscles: role of endocardial endothelium. CircRes 69: 301–312.of calcium on force-velocity-length relations of heart

muscle of the cat. Circ Res 32: 385–392. L K, X SM, 1989. The role of endothelial cells onvascular relaxation. Adv Cardiovasc Dis 10: 16–20.B DL, M AL, S KR, S SU, 1988.

Effects of damaging the endocardial surface on the M F, B P, L C, P E, P- G, G JH, H TE, 1990. Contractile effectmechanical performance of isolated cardiac muscle.

Circ Res 62: 358–366. of the chemotactic factor f-Mef-Leu-Phe and C5a onthe human isolated umbilical artery: role of cyclo-C W, 1991. Smooth muscle cell growth factors.

Prog Growth Factor Res 3: 177–206. oxygenase products and tissue macrophages. Circ Res67: 1059–1070.C P, S DJ, 1989. Immunoreactive en-

dothelin in human plasma: marked elevations in M AL, S KR, S SU, B DL, 1988.Atriopeptin III induces early relaxation of isolatedpatients in cardiogenic shock. Biochem Biophys Res

Commun 161: 562–567. mammalian papillary muscle. Circ Res 62: 1171–1174.N BJ, 1972. The effects of indomethacin onD’O C, G L, Q D, R A, 1992.

The dependency of collagen fibrillogenesis in vitro on calcium, sodium, potassium and magnesium fluxes invarious tissues of the guinea pig. Br J Pharmacol 45:fibroblast culture conditions. Fibroblasts in mono- and

multi-layers. Arch Histol Cytol 55: 235–241. 651–659.N BJ, 1973. The effects of indomethacin onE M, B JR, 1988. Actions of sympathomimetic

amines on the Ca2+ transients and contractions of calcium, potassium and magnesium fluxes in varioustissues of the guinea pig. Br J Pharmacol 48: 496–504.rabbit myocardium: reciprocal changes in myofibrillar

responsiveness to Ca2+-mediated through a- and b- R MA, M T, N M, B M, W- S, W J, 1993. Effect of aspirin on theadrenoceptors. Circ Res 62: 247–265.

E M, H T, I A, T M, I contractility of aortic rings in vitro from spontaneouslyhypersensitive rats. Artery 20: 135–146.J, 1991. Myocardial a1-adrenoceptors mediate positive

inotropic effect and changes in phosphatidylinositol R C, MC G, S A, R D, W- A, W S, 1993. Cardiac endothelial cellsmetabolism: species differences in receptor distribution

and the intracellular coupling process in mammalian modulate contractility of rat heart in response to oxy-gen tension and coronary flow. Circ Res 72: 1044–ventricular myocardium. Circ Res 68: 1179–1190.

F RF, Z JV, 1980. The obligatory role 1064.R C, S A, MC G, W S,of endothelial cells in the relaxation of arterial smooth

muscle by acetylcholine. Nature 288: 373–376. 1992. Endothelial cells regulate cardiac contractility.Proc Natl Acad Sci USA 89: 4033–4036.J JY, M DP, D P, 1990. Differential

inhibitory potencies of non-steroidal antiinflammatory S VB, C S, S-U B, B R, V- PM, 1994. Insulin-like growth factor I inhibitsdrugs on smooth muscle prostnoid synthesis. Eur J

Pharmacol 182: 83–89. induction of nitric oxide synthase in vascular smoothmuscle cells. Circ Res 74: 24–32.J A, R J, R L, 1991. Effect of dextra on

synthesis, secretion and deposition of type III pro- S K, H T, 1992. Inotropic actions of eico-sanoids. Basic Res Cardiol 87: 2–11.collagen in cultured human fibroblasts. Biochem J 279:

49–54. S-B T, R TJ, H AW, V PM,1991. Induction of endothelin secretion by angiotensinK H, W B, S Y, H G, V B,

G A, 1993. Procollage types I and III and II: effects on growth and synthetic activity of vascularsmooth muscle cells. J Cardiovasc Pharmacol 17 [Suppl.transforming growth factor-beta gene expression in

the arterial wall after exposure to periarterial blood. 7]: S96–S100.S AM, A LJ, M AL, B DL,Neurosurgery 33: 716–721.

K M, N Y, M S, S K, K Y, 1989a. Endocardium modulates myocardial inotropicresponse to 5-hydroxytryptamine. Am J Physiol 257:1994. Atrial natriuretic factor and isosorbide dinitrate

modulate the gating of ATP-sensitive K+ channels in H1790–H1787.S AM, M AL, B DL, 1989b. Myo-cultured vascular smooth muscle cells. Circ Res 74:

471–476. cardial inotropic responses to aggregating platelet andmodulation by the endocardium. Circulation 79: 1315–L K, C X, 1987. Effect of captopril and enalapril on

myocardial ischemia and reperfusion damage in rat. 1323.S H, 1990. Mechanisms of release and renalJ Mol Cell Cardiol 19: 909–915.

L K, R JL, 1991. a1-adrenergic stimulation in- action of atrial natriuretic factor. Acta Physiol Scand591: 80–87.creases the Vmax of isolated myocardial papillary

muscles. Can J Physiol Pharmacol 69: 1804–1809. S DJ, L D, C P, C K,1990. Endothelin release is inhibited by coculture ofL K, R JL, A L, B DL, 1993a. Effect

of dysfunctional vascular endothelium on myocardial endothelial cells with cells of vascular media. Am JPhysiol 259: H1928–H1932.performance in isolated papillary muscles. Circ Res 72:

768–777. S B, S G, -J L, S- K- C, 1993. Matrix production of smooth muscleL K, R JL, C A, A L, B

DL, 1993a. Endocardial dysfunction in pacing-induced cells from rat aorta in vitro. Histol Histopathol 8: 63–72.T-O M, T Z, K K, 1990. En-heart failure in the dog. J Mol Cell Cardiol 25: 529–540.

L K, S P, R JL, 1994. Role of endothelial cells dothelin-1 and platelet activating factor stimulatethromboxane A2 biosynthesis in rat vascular smoothin cardiovascular function. Life Science 54: 579–592.

L K, S DJ, R JL, 1991. Myocardial con- muscle cells. Biochem Pharmacol 40: 2713–2717.T PR, C K, H T, S AG, 1994.tractile actions of endothelin-1 in rat and rabbit

K. Li et al.892

Platelet-activating factor biosynthesis in rat vascular W J, P G, M JP, 1991. Endothelin 1 enhancesmyofilament Ca2+ responsiveness in aequorin-loadedsmooth muscle cells. J Vasc Res 31: 144–152.

V PM, L TF, G T, 1991. Endothelin- ferret myocardium. Circ Res 69: 582–589.Y M, K H, K S, T Y, K-dependent contractions. Blood Vessels 28: 74–83.

W J, M JP, 1992. Endocardial endothelium mod- M, M Y, Y Y, G K, M T, 1988.A novel potent vasoconstrictor peptide produced by vas-ulates myofilament Ca2+ responsiveness in aequorin-

loaded ferret myocardium. Circ Res 70: 754–760. cular endothelial cells. Nature 332: 411–415.