mechanism utilizes the PI3-K/PKB signal transduction

pathway.

doi:10.1016/j.yjmcc.2006.06.039

26. Tissue Engineering of Spontaneously Beating,

Vascularised, Three-Dimensional Cardiac Tissue

A.N. Morritt*,1, R.J. Dilley 2, J. Rickards 2, X.L. Han 1, D.

McCombe 1, W.A. Morrison 1, S.K. Bortolotto 1. 1 The

Bernard O’Brien Institute of Microsurgery, Melbourne. 2 The

Department of Medicine, University of Melbourne, Melbourne,

Vic, Australia

Engineered cardiac tissue could be used clinically to repair

congenital or acquired cardiac defects, or experimentally to

assess the efficacy and safety of new drugs. The aim of this

study was to engineer vascularised three-dimensional cardiac

tissue in vivo.

Methods: Neonatal rat cardiomyocytes were isolated,

cultured, seeded inMatrigel, and placed around an arteriovenous

blood vessel loop within a polycarbonate chamber in the groin of

an adult nude rat. Chambers were harvested up to 10 weeks later,

and some specimens underwent functional testing. All tissue was

fixed, embedded in paraffin, sections cut and stained for DAPI,

H and E, Massons trichrome and immunostained for Desmin,

alpha sarcomeric actin, Connexin-43, Ki67 and Lectin.

Results: All tissue harvested at 1 week (n =4), 4 weeks

(n =8), 5 weeks (n =2) and 10 weeks (n =5) contained

cardiomyocytes on staining with desmin and alpha sarcomeric

actin, and was profusely vascularised on staining with H and E

and Lectin. All tissue constructs harvested after 1 week were

observed to beat spontaneously. Connexin-43 immunostain

identified gap junctions between cardiomyocytes, and Ki67

staining showed that some cardiomyocytes were still dividing at

4 weeks. Tissue harvested at 4 and 10 weeks exhibited a positive

chronotropic response to noradenaline, a positive inotropic

response to calcium, and could be paced electrically at various

rates without tetany.

Conclusion: Vascularised, spontaneously beating, three-

dimensional cardiac tissue can be grown by placing cardio-

myocytes from neonatal rat hearts within a rat tissue

engineering chamber.

doi:10.1016/j.yjmcc.2006.06.040

27. Wave intensity analysis (WIA): flow velocity lags

influence calculated ascending aortic wave speed and

reflected wave intensity during beta-adrenergic stimulation

Jonathan P. Mynard*, Joseph J. Smolich, Daniel J. Penny.

Australia and New Zealand Children’s Heart Research Centre,

Murdoch Children’s Research Institute, Parkville, Victoria,

Australia

With WIA, a powerful new technique for assessing

ventriculo-arterial interaction, the intensity of vascular energy

waves is obtained as the instantaneous product of pressure and

flow velocity changes, and can be separated into components

propagated from the heart and reflected from the vasculature.

Wave separation requires calculation of wave speed, which can

be confounded by hardware-related time lags in pressure-

velocity signals. Although wave separation is relatively

insensitive to lag-related wave speed variations under baseline

conditions, the effect of such lags on wave speed or wave

separation during beta-adrenergic stimulation is unknown.

To address this question, 10 anaesthetized, open-chest adult

sheep were instrumented with an ascending aortic ultrasonic

flow probe and micromanometer-tipped catheter. Pressure-

velocity data were acquired at 500 Hz at baseline and after

infusion of 2.5, 5 and 10 Ag/min/kg of dobutamine. Using

WIA, wave speed and separated wave intensities were

determined with pressure-velocity data aligned using the peak

second-derivatives of pressure and velocity in early systole

(‘‘zero-lag’’), and with applied velocity or pressure lags of one

or two sample intervals (2 ms).

Wave speed and wave separation were not significantly

affected by pressure or velocity lags at baseline, or by pressure

lags during dobutamine infusion. However velocity lags

progressively increased wave speed (0.001<p <0.04) and

reflected wave intensities (0.001<p <0.03) during incremental

dobutamine infusion, sometimes even resulting in the appear-

ance of reflected waves not present at zero-lag.

Conclusion: Use of WIA in the setting of beta-adrenergic

stimulation requires accurate pressure-velocity signal align-

ment to prevent errors in calculated wave speed and reflected

wave intensity.

doi:10.1016/j.yjmcc.2006.06.041

28. Ovariectomy exacerbates the hypertrophic response in

a mouse model of primary cardiac hypertrophy

Ruchi Patel*,1, Rebecca H. Ritchie 2, Claire L. Curl 3, Lea M.

Delbridge 3, Igor R. Wendt 1. 1 Department of Physiology,

Monash University. 2 Baker Heart Research Institute.3 Department of Physiology, Melbourne University,

Melbourne, Australia

There is evidence for sex-based differences in the develop-

ment of cardiac hypertrophy that cannot be explained by

differences in blood pressure. As functional oestrogen and

testosterone receptors have been identified in the heart it is

likely that sex-steroids may be responsible for such male-

female differences. A transgenic mouse model, in which

cardiac hypertrophy is driven by cardiac-specific overexpres-

sion of angiotensinogen, independent of changes in blood

pressure (TG 1306), was used to investigate sex differences and

the roles of the sex hormones in heart growth. We hypothesised

that absence of oestrogen and presence of testosterone both

exert pro-growth influences in the heart and, where there is

genetic predisposition for cardiac hypertrophy, these effects are

more pronounced. Male and female wild type (WT) and

transgenic (TG) mice underwent gonadectomy (GDX) at 12

Abstracts / Journal of Molecular and Cellular Cardiology 41 (2006) 732–751742

weeks of age. 4 weeks post surgery body weight and blood

pressure were recorded and the hearts collected for determina-

tion of cardiac and ventricular weight indices (CWI and VWI

mg/g). GDX had no effect on heart weight in WT or TG male

mice compared to sham-operated males. In females GDX had

no effect on heart weight in WT mice, however, tended to

increase heart weight in TG mice (n =5) compared to sham-

operated TG controls (n =7) (CWI 6.4T0.62 GDX vs

5.66T0.19 sham and VWI 6.05T0.59 GDX vs 5.32T0.17sham). These results suggest that the absence of oestrogen

exacerbates heart growth where there is genetic predisposition

for cardiac hypertrophy.

doi:10.1016/j.yjmcc.2006.06.042

29. Cardioprotection in aged myocardium: Sites of failure

in protective signalling

Jason Peart*, Melissa E. Reichelt, Laura Willems, John

Headrick. Heart Foundation Research Centre, Griffith

University, Australia

Ischaemic tolerance and cardiac protection appear altered in

a detrimental manner with ageing. Our prior work supports

altered protective signalling distal to adenosine and other G-

protein coupled receptors. Here we investigated abilities of

pharmacological modulation of specific signalling intermedi-

ates (implicated in cardioprotection) to ameliorate injury in

young and aged myocardium. Langendorff perfused mouse

hearts (young- 2–4 mth; aged- 18 mth) were subjected to 20–

25 min global ischaemia and 45 min normoxic reperfusion.

Pre-ischaemic function was comparable in all experimental

groups. Young hearts recovered ¨50% of contractile function

(71T5 mmHg developed pressure), with significant diastolic

contracture (26T4 mmHg) and efflux of LDH (14T3 IU/g;

reflecting cell death). Ageing was associated with substantially

impaired functional outcome (38T3 mmHg developed pres-

sure) and greater extent of necrosis (26T4 IU/g). Acute

adenosine treatment, morphine treatment, and ischaemic

preconditioning (2�5 min ischaemic stimuli) all enhanced

ischaemic tolerance in young but not aged hearts. Ageing also

abrogated phorbol 12-myristate 13-acetate (PMA) mediated

(PKC-dependent) cardioprotection. However, activation of

mitochondrial ATP-sensitive K+ channels (50 AM diazoxide)

and inhibition of the mitochondrial permeability transition pore

(MPT; with 0.3 AM cyclosporin A) protected both young and

aged hearts. Activation of p38 MAPK with anisomycin was

also protective in young and aged hearts. These data show that

multiple protective stimuli effective in young myocardium

(adenosine, opioids, preconditioning) are ineffective in aged

hearts. Failed protection appears to occur distal to receptors and

PKC, proximal to mitochondrial targets (ATP-sensitive K+

channels, the MPT), and potentially at the level of p38 MAPK

signalling.

doi:10.1016/j.yjmcc.2006.06.043

30. p38 MAPK activation restores cardioprotection to the

aged heart

J. Peart*,1, G. Gross 2, J. Headrick 1. 1 Heart Foundation

Research Centre, Griffith University, Australia. 2 Medical

College of Wisconsin, USA

Preconditioning mediated via G-protein coupled receptors is

typically lost with advancing age. We report untreated hearts

from young (3 months) and aged C57/Bl6 mice (18 months)

recovered similarly following ischemia and reperfusion.

However, treatment with the delta-opioid receptor ligand,

DPDPE (1 AM), significantly improved recovery in young,

but failed to alter recovery in aged hearts. Examination of post-

receptor signalling through Western immunoblot analysis

following acute opioid receptor activation revealed similar

translocation of GRK2 along with phosphorylation of Akt,

p42/44 MAPK and p70S6K in both young and aged hearts.

However, while DPDPE stimulation produced a pronounced

phosphorylation of p38 MAPK and Heat Shock Protein 27

(HSP27), a mediator of p38 MAPK, in the young hearts,

phosphorylation was unchanged in aged hearts.

Based on these findings, we investigated the role of p38

MAPK in the age-related loss of opioid preconditioning.

Langendorff-perfused hearts from young or aged C57/Bl6

mice underwent 25 min ischemia and 45 min reperfusion in

the presence or absence of 1 AM anisomycin, an activator of

p38 MAPK. Contractile recovery was paralleled in untreated

young and aged hearts (50T1% and 53T5%, respectively).

Importantly, anisomycin treatment elicited pronounced phos-

phorylation of both p38 MAPK and Hsp27, while affording

significant anti-ischemic protection, comparable in both

young and aged hearts (73T3% and 76T2%, respectively.

p <0.05 vs untreated).

In summary, while anti-stunning effect of delta-opioid

receptor activation is lost in the aged heart, exogenous

activation of p38 MAPK maintains preconditioning in the

aged. These results suggest a failure in post-receptor signalling

upstream of p38 MAPK.

doi:10.1016/j.yjmcc.2006.06.044

31. Trigger and mediation phase of chronic opioid

preconditioning are mediated via divergent pathways

J. Peart*,1, J. Headrick 1, G. Gross 2. 1 Heart Foundation

Research Centre, Griffith University, Australia. 2 Medical

College of Wisconsin, USA

Chronic opioid preconditioning confers a pronounced and

prolonged cardioprotective phenotype, existing at least 24 hours

beyond opioid drug withdrawal. This study aims to delineate

both the trigger and mediation phase of this cardioprotective

state. To this end, we employed an isolated, perfused murine

heart model of ischemia reperfusion. Following 25min ischemia

and 45 min reperfusion, placebo-treated hearts exhibited a

pronounced degree of contractile dysfunction (EDP, 32T3mmHg; RPP, 40T4% baseline). In stark contrast, chronic

Abstracts / Journal of Molecular and Cellular Cardiology 41 (2006) 732–751 743