S362 Canadian Journal of CardiologyVolume 29 2013

myocardial intracellular calcium handling due to reducedsarcoplasmic reticulum ATPase (SERCA2a) expression andfunction. Exercise-training is known to counteract these effectsand restores SERCA2a function in obese mice. However,a common mechanism to explain how high-fat feeding andexercise influences SERCA2a function in the heart has not yetbeen identified. AMPK (5' adenosine monophosphate-acti-vated protein kinase) senses fluctuations in cellular energyhomeostasis during metabolic stress and physical activity.Therefore, we tested the hypothesis that AMPK influencesmyocardial SERCA2a protein content and activity in the heart.METHODS: Wild-type (WT) and AMPKa2 kinase-dead (KD)transgenic mice (which is a genetic manipulation that reducesAMPK activity by w70%, as compared to WT mice) werefed a standard rodent chow (low fat diet; LFD) or a 40% highfat diet (HFD) for 5 months. Half of the LFD and HFD micewere provided access to voluntary running wheels (+EX),whereas the remainder were housed in standard housing andserved as the sedentary reference group (+SED). Our decisionto utilize these models was based on the fact that high fatfeeding and sedentary behaviour down-regulates the activationof AMPK; whereas, exercise-training enhances AMPK activityin standard chow fed or high fat fed mice.RESULTS: KDmice, as compared toWT, were characterized byreduced SERCA2a and higher phospholamban (PLN) proteinlevels in tissue isolated from the left ventricular. Although heartweights did not differ betweenWT and KDmice, heart weightsamongst the HFD+SED mice were increased by 41%, ascompared to LFD+SED, which indicates that high fat feedinginduced hypertrophy. The high-fat diet also induced diastolicdysfunction, as indicated by a prolonged deceleration timeassessed using tissue Doppler imaging. SERCA2a proteincontent was also reduced by 36% amongst HFD+SED, ascompared to LFD+SED. Exercise up-regulated myocardialSERCA2a protein content by 43% amongst WT mice in theLFD+Ex group, as compared to WT mice in the LFD+Sedgroup. Exercise also prevented the development of cardiacdysfunction and the down-regulation of SERCA2a mRNA andprotein content amongst theWTmice fed aHFD, as comparedto the HFD+SED group. However, exercise-training did notalter myocardial SERCA2a protein content amongst KD mice.CONCLUSION: Based on these data, we suggest that AMPKinfluences myocardial SERCA2a expression and function inresponse to high-fat feeding and exercise training.CIHR

693BASIC FIBROBLAST GROWTH FACTOR SUPPRESSESTRANSFORMING GROWTH FACTOR-b1-MEDIATED HUMANCARDIAC FIBROBLAST DIFFERENTIATION INTOMYOFIBROBLASTS

JM Ngu, G Teng, BD Lipon, HE Mewhort, CK Seneviratne,PW Fedak

Calgary, Alberta

BACKGROUND: After myocardial injury, sustained elevation ofpro-fibrotic cytokine transforming growth factor-beta1 (TGF-b1) activates cardiac myofibroblasts resulting in fibrosis andheart failure. Basic fibroblast growth factor (bFGF) inhibitsTGF-b1-mediated myofibroblast activation in 2D cultureexperiments. In this study, we uniquely examined the effectsof bFGF on TGF-b1-stimulated human cardiac fibroblasts in3D collagen matrices.METHODS AND RESULTS: Cardiac fibroblasts were isolatedfrom human right appendage tissues and left ventricular corebiopsies obtained from cardiac surgery patients (N¼8).Cultured cardiac fibroblasts were seeded in 3D collagen gels,which contract in proportion to the extent of differentiationinto myofibroblasts. Cells were treated with serum-freemedium (SFM), either alone or containing TGF-b1 (10 ng/mL) or TGF-b1 (10 ng/mL) + bFGF (20 ng/mL). Gels wereanalyzed for degree of contraction, alpha-smooth muscle actin(a-SMA) expression, collagen density, and microRNA profile.TGF-b1 significantly increased the baseline gel contraction(76.3�1.6% [TGF-b1] vs. 66.6�3.1% [SFM], P¼0.03),reflecting an activation of myofibroblasts. bFGF inhibited theTGF-b1-mediated gel contraction (65.2�6.6%[bFGF+TGF-b1] vs. 76.3�1.6% [TGF-b1], P¼0.01).Immunocytochemistry staining with a-SMA (a phenotypicmarker of differentiated myofibroblasts) showed that bFGFreduced the percentage of a-SMA expressing cells whencompared to TGF-b1 (74.7�5.4% [bFGF+TGF-b1] vs86.5�5.6% [TGF-b1], P¼0.0006). Flow cytometryconfirmed that bFGF suppressed the cellular a-SMA expres-sion triggered by TGF-b1 (18900 [bFGF+TGF-b1] vs.21000 [TGF-b1], arbitrary units). 3D confocal microscopicimages of the collagen gel matrix demonstrated that TGF-b1increased collagen density while bFGF significantly attenuatedgel fibrosis.

Abstracts S363

The microRNA (miRNA) expression profile wasexamined using a high throughput microarray platform.TGF-b1 induced several pro-fibrotic miRNAs whencompared to the SFM control: miRNA-10a (2.01-fold),miRNA-127-5p (3.25-fold), miRNA-192 (1.89-fold) andmiRNA-346 (1.64-fold). Addition of bFGF significantlyreduced these pro-fibrotic miRNAs: miRNA-10a (1.52-fold), miRNA-127-5p (1.59 fold), miRNA-192 (2.71-fold)and miRNA-346 (1.70-fold). Human ventricular cardiacfibroblasts were employed to determine whether these cellsrespond similarly to atrial derived fibroblasts: TGF-b1stimulated the collagen gel contraction (53.2�3.2% [TGF-b1] vs. 31.3�4.0% [SFM], P<0.0001) and the additionof bFGF abolished the TGF-b1-induced gel contraction(35.5�1.4% [bFGF+TGF-b1] vs 53.2�3.2% [TGF-b1],P<0.0001).CONCLUSION: In summary, these data demonstrate thatbFGF suppresses TGF-b1-mediated human cardiac fibro-blast differentiation into myofibroblasts and preventsmyocardial fibrosis. Ventricular and atrial human cardiacfibroblasts respond similarly to bFGF therapy. bFGF isa promising therapeutic agent to prevent maladaptivestructural remodeling and heart failure following myocardialinjury.

694a11 INTEGRIN DEFICIENCY IN MICE IS ASSOCIATED WITHIMPAIRED DIASTOLIC FUNCTION AND MYOFIBRILLARDISARRAY

KA Connelly, I Talior-Volodarsky, M Mitchell, J Desjardins,G Kabir, C McCulloch

Toronto, Ontario

BACKGROUND: The family of Integrins are transmembranereceptors that have two major functions; attachment of thecell to the extracellular matrix (ECM) and signal transductionfrom the ECM to the cell. As a result they play crucial roles indiverse cellular and developmental processes including cellgrowth, differentiation and survival. We have previouslydemonstrated that diabetes induces the cardiac expression ofthe a11 integrin that, via Smad dependent mechanismsmediates the formation of profibrotic fibroblasts andcontributes to the development of a fibrotic interstitium. Theexact role of a11 integrin in cardiovascular developmenthowever remains obscure. We therefore utilized the a11integrin KO mouse to identify the role of endogenous a11integrin in the myocardium.METHODS AND RESULTS: As lack of a11 integrin results inloss of the periodontal ligament, a11 integrin mice werefed mashed food to ensure normal growth. At 2 months ofage, a11 integrin KO mice and wildtype littermatesunderwent echocardiography, invasive pressure volumeloop analysis followed by euthanasia with tissue collectionfor histology.

By 2 months a11 integrin KO mice demonstrated noobvious phenotype. Body weight was mildly reduced C/wlittermate controls (38 �3g vs 32 � 3g, p<0.01). Tibiallength (TL) was not different (14 � 0.4mm vs 14 � 0.8mm,p¼0.28). Height weight indexed to TL was also not different(10.4 � 0.7 vs 10.3 � 1.7 mg/mm, p¼0.6). Lung weightindexed to TL was increased, suggesting pulmonary conges-tion (14 �2 vs 11 � 2, g/mm p<0.05). Echocardiographydemonstrated preserved systolic function and no evidence forLV remodeling. Invasive PV loop analysis demonstratedimpaired diastolic function in a11 integrin KO mice mani-festing as increased end diastolic pressure, prolonged Tau aswell as steeper end diastolic pressure volume relationships (allp<0.05), despite no difference in heart rate or systolic bloodpressure. Functional changes were associated with structuralalterations. a11 integrin KO mice demonstrated a reductionin cardiomyocyte width (p<0.05 c/w WT), along withmyofibrillar disarray (figure 1, wheat germ agglutinin (WGA)staining to outline cardiomyocytes) and increased extracellularmatrix deposition.CONCLUSION: Loss of the a11 integrin in 2 month old micewas associated with impaired diastolic function and prominentmyofibrillar disarray. These findings suggest that a11 integrinplays a vital role in cardiovascular development. Furtherstudies to determine the mechanism and potential clinicalsignificance are warranted.

HSFC

695THE ROLE OF TISSUE INHIBITOR OF METALLOPROTEINASE-2 INHUMAN CARDIAC FIBROBLAST-MEDIATED EXTRACELLULARMATRIX REMODELING

JM Ngu, CH Meijndert, HE Mewhort, JD Turnbull, WV Yong,WG Stetler-Stevenson, PW Fedak

Calgary, Alberta

BACKGROUND: Tissue Inhibitor of Metalloproteinase-2(TIMP-2) is an endogenous inhibitor of matrix-