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Metabolic phenotyping of the diseased heart using 13C-substrates and Ex vivo perfusion in the working mode
ADENOSINE KINASE INHIBITOR (AKI) MODULATION OF POST-MYOCARDIAL INFARCTION (Ml) FIBROSIS Francisco Villarreal, Lala Makhsudova, Sara Jeffrey Omens, Bruce Ito. Dept of Medicine Metabasis Therapeutics, Inc.
Epperson, UCSD and
Adenosine (ADO) inhibits cardiac fibroblast function. Enhanced ADO levels in the setting of Ml may inhibit scarring/fibrosis and ameliorate ventricular remodeling. AKI increase tissue ADO levels. Studies were performed in rats to assess the effects of AKI administered 48 h after Ml on LV scarring and remodeling. Treatment was provided as either vehicle (control group) or the AKI agent GP515 (5 mglkglday) using a miniosmotic pump 48 h after MI. Studies were performed 14 days after Ml. Results indicated similar MI sizes (3425% vs. 39_8% AKI) or heart weight to body weight ratios (4.050.2 vs. 4.4_0.3 AKI). AKI treatment increased infarcted (1.820.4 vs. 1.2_+0.4 mm, p=O.O2) and septal wall thicknesses (2.930.7 vs. 2.3+0.4 mm). Pressure volume relationships showed a right shrftwith AKI treatment with no differences in LVvolumes at Vo. Scar collagen area fraction in untreated rats yielded 7&+9 % vs. 53+8 % with AKI (p-=0.05). Passive circumferential segment lengthening at 15 mml-lg was greater for AKI (0.13_0.05 vs. 0.07+0.06, p
REMODELLING OF THE SARCOLEMYA IN DIABETIC (DIA) RAT HEARTS: THE ROLE OF MEMBRANE FLUIDITY lveta Waczuliiv& Libulla &kurov#, Jozef &rsky~, Barbara Ziegelh6ffer - Mlhabvi&v#, Atttla Ziegelh6fier~ IDept Biophys, Fat Natr Sd & 4nst Med Chem Bbchsm, Fat led, ComeniusUniv, %st Heatt Res,SlovakAcad Sd, Bratislava,Sbvakia DIA hearts
exhibit rsduced
(by 4763
%) sensitivity
to Ca-overloacl.
This is based on rerncdeling of the safcclemmal membranes (SL) with down-regulated, but kinetically well preserved Na,K-ATPase and abnormal Mg/Ca -ATPaw (all p
(streptozotocin,single dose, 45 mgkg ix) were treated in a special regimen with resorcylidene aminoguanidine (RAG, 4 mgkg i.m.) The treatmentwith RAG eliminated completely the DIA-induced decrease in the SLMF [p < 0.001) as well as in NEG, and FR in the SL (p < 0.05), but it influenced only littta the SL ATPasa -activities both, in healthy and DIA animals (p > 0.05). On the other hand, it increased considerably the vulnerability of the heart to overload with external Ca. Conclusions: i) In DIA hearts, the alterations in SLMF(induosd by NEG and FRF) hardly may bs oonsidered as a reason for changes in the ATPases-mediated processes in SL; ii) The NEG and FRF caused alterations in SLMF seem to represent a part of adaptation changes protecting the DIA heart against an overload by Ca. Supported by VEGA grants No. 2/7157/20 and li7673120, and by Slovak Ministry of Education grant No. 116145199.
Al26
METABOLIC PHENOTYPING OF THE DISEASED HEART USING “C-SUBSTRATES AND EX V/V0 PERFUSION IN THE WORKING MODE. Genevieve Vincent’, Maya Khalrallah, Bertrand Bouchard, Joanne K. Kelleher & Chrlstine Des Rosiers, Dspts Biochemistry & Nutrltion, CHUM Res. Ctr, Univ. Monttial, Canada; Dept Physlology, GW Univ., USA. Although accelerated glycolysis is shown to be part of the metabolic phenotype expressed by the hypertrophied heart, less is known about the impact of hypertrophy on the activity of mitochondrial citric acid cycle (CAC) reactions. Methods: We have used 13C-mass isotopomer analysis by gas chromatography-mass spectrometry to assess pyruvate partitioning between decarboxylation (PDC) and carboxylation (PC) for mitochondrial citrate formation and efflux (reflected by citrate release rate) in spontaneously hypertensive rat (SHR: 15 week-old) hearts. Wistar-Kyoto (WKY) rats served as controls. Working hearts were perfused with 5.5 mM glucose, 8 nM insulin, 1 mM ‘3C,lactate (Lac), 0.2 mM “C3-pyruvate (Pyr), and 50 uM camitine. Results: Compared to WKY, SHR hearts showed significantly lower left ventricular contractility, cardiac output, aottic and coronary flows, cardiac power (CP) and efficiency. This mechanical dysfunction was associated with the following metabolic perturbations (normalised to CP): an increase of [Lac + Pyr] and citrate release rates, and a decrease of [Lac + Pyr] uptake, PDClPC and PDC/CAC flux Since SHR hearts ratios and CAC flux rate. Conclusion: release more citrate, suggesting that substrate supply for citrate synthesis exceeds citrate utilisation in the CAC, mitochondrial pyruvate oxidation appears to be restricted beyond the citrate synthase reaction. (Funded by the CIHR).
Cd+ WAVES FROM THE BORDER ZONE WITH NONUNIFORM EXCITATION-CONTRACTION COUPLINQ Yuji Wakayame, Bturm DM Stuyvefs, Henk EDJ tar Keurr Dept of Medllinr. University of Calgary, Calgary. Alberta. Cmmde. Triggered propagated contractkns (TPCs) and underlying Ce” waves ere initiated from local dameged regkne in cardlec muscle end cause enhythmlas. In order to study whether nonunIform extdetiincontraction coupling (ECC) ceueee TPCe and Ca* waves, we prepared 11 ret trebsculse with nonunlfam ECC created by local exposure to a smell jet of sallne solution Including 5mM caffeine (CAF) or 2CmM P.S-butanedkne monoxime (BDM). me jet wee applied perpendicularly to the muscle from a glass pipMe (-1 OOpm tip) at a constant flow (O.OSml!min). Force was measured with a etlkon strain gauge. sarcomsre length with laser diffraction techniquas and [Co?, with eleotrophoretiially injected Furs-2 end en image Intenslfied CCD camera. In the region where the trabecula wee exposed to CAF or BDM (-6Wpm). sarcomerss reversibly etrekhed during the twitch, while outside the jet solutkn they continued to shorten, Indketlng locally decreased ECC. Car wavse could be lnducsd, in a controlled manner, from the border between shortening and stretched rsgions following 2.5Hz stimulus trains (75sec, 23-C. [Ce’\=Z.OmM). Ce” waves invariably started late during relaxation of the last stimulated twitch, corresponding to 814% (CAF; n=14) and SS&% (BDM; n=S) relaxation of active fcme. and then propegeted toward the normal regions at velocbies of 0.75+.0.10 (CM) end 0.B540.14mm/eec Arrhythmias also could be induced using this method. These rssulte show that nonuniform ECC, lrraepalve of local damage, can cause Ca” waves end arrhythmlee and that Ca* dksodated from myofllaments during relexatkn in the border xons may play en Important role in the Initiation c Cab waves I
Epperson, UCSD and
Adenosine (ADO) inhibits cardiac fibroblast function. Enhanced ADO levels in the setting of Ml may inhibit scarring/fibrosis and ameliorate ventricular remodeling. AKI increase tissue ADO levels. Studies were performed in rats to assess the effects of AKI administered 48 h after Ml on LV scarring and remodeling. Treatment was provided as either vehicle (control group) or the AKI agent GP515 (5 mglkglday) using a miniosmotic pump 48 h after MI. Studies were performed 14 days after Ml. Results indicated similar MI sizes (3425% vs. 39_8% AKI) or heart weight to body weight ratios (4.050.2 vs. 4.4_0.3 AKI). AKI treatment increased infarcted (1.820.4 vs. 1.2_+0.4 mm, p=O.O2) and septal wall thicknesses (2.930.7 vs. 2.3+0.4 mm). Pressure volume relationships showed a right shrftwith AKI treatment with no differences in LVvolumes at Vo. Scar collagen area fraction in untreated rats yielded 7&+9 % vs. 53+8 % with AKI (p-=0.05). Passive circumferential segment lengthening at 15 mml-lg was greater for AKI (0.13_0.05 vs. 0.07+0.06, p
REMODELLING OF THE SARCOLEMYA IN DIABETIC (DIA) RAT HEARTS: THE ROLE OF MEMBRANE FLUIDITY lveta Waczuliiv& Libulla &kurov#, Jozef &rsky~, Barbara Ziegelh6ffer - Mlhabvi&v#, Atttla Ziegelh6fier~ IDept Biophys, Fat Natr Sd & 4nst Med Chem Bbchsm, Fat led, ComeniusUniv, %st Heatt Res,SlovakAcad Sd, Bratislava,Sbvakia DIA hearts
exhibit rsduced
(by 4763
%) sensitivity
to Ca-overloacl.
This is based on rerncdeling of the safcclemmal membranes (SL) with down-regulated, but kinetically well preserved Na,K-ATPase and abnormal Mg/Ca -ATPaw (all p
(streptozotocin,single dose, 45 mgkg ix) were treated in a special regimen with resorcylidene aminoguanidine (RAG, 4 mgkg i.m.) The treatmentwith RAG eliminated completely the DIA-induced decrease in the SLMF [p < 0.001) as well as in NEG, and FR in the SL (p < 0.05), but it influenced only littta the SL ATPasa -activities both, in healthy and DIA animals (p > 0.05). On the other hand, it increased considerably the vulnerability of the heart to overload with external Ca. Conclusions: i) In DIA hearts, the alterations in SLMF(induosd by NEG and FRF) hardly may bs oonsidered as a reason for changes in the ATPases-mediated processes in SL; ii) The NEG and FRF caused alterations in SLMF seem to represent a part of adaptation changes protecting the DIA heart against an overload by Ca. Supported by VEGA grants No. 2/7157/20 and li7673120, and by Slovak Ministry of Education grant No. 116145199.
Al26
METABOLIC PHENOTYPING OF THE DISEASED HEART USING “C-SUBSTRATES AND EX V/V0 PERFUSION IN THE WORKING MODE. Genevieve Vincent’, Maya Khalrallah, Bertrand Bouchard, Joanne K. Kelleher & Chrlstine Des Rosiers, Dspts Biochemistry & Nutrltion, CHUM Res. Ctr, Univ. Monttial, Canada; Dept Physlology, GW Univ., USA. Although accelerated glycolysis is shown to be part of the metabolic phenotype expressed by the hypertrophied heart, less is known about the impact of hypertrophy on the activity of mitochondrial citric acid cycle (CAC) reactions. Methods: We have used 13C-mass isotopomer analysis by gas chromatography-mass spectrometry to assess pyruvate partitioning between decarboxylation (PDC) and carboxylation (PC) for mitochondrial citrate formation and efflux (reflected by citrate release rate) in spontaneously hypertensive rat (SHR: 15 week-old) hearts. Wistar-Kyoto (WKY) rats served as controls. Working hearts were perfused with 5.5 mM glucose, 8 nM insulin, 1 mM ‘3C,lactate (Lac), 0.2 mM “C3-pyruvate (Pyr), and 50 uM camitine. Results: Compared to WKY, SHR hearts showed significantly lower left ventricular contractility, cardiac output, aottic and coronary flows, cardiac power (CP) and efficiency. This mechanical dysfunction was associated with the following metabolic perturbations (normalised to CP): an increase of [Lac + Pyr] and citrate release rates, and a decrease of [Lac + Pyr] uptake, PDClPC and PDC/CAC flux Since SHR hearts ratios and CAC flux rate. Conclusion: release more citrate, suggesting that substrate supply for citrate synthesis exceeds citrate utilisation in the CAC, mitochondrial pyruvate oxidation appears to be restricted beyond the citrate synthase reaction. (Funded by the CIHR).
Cd+ WAVES FROM THE BORDER ZONE WITH NONUNIFORM EXCITATION-CONTRACTION COUPLINQ Yuji Wakayame, Bturm DM Stuyvefs, Henk EDJ tar Keurr Dept of Medllinr. University of Calgary, Calgary. Alberta. Cmmde. Triggered propagated contractkns (TPCs) and underlying Ce” waves ere initiated from local dameged regkne in cardlec muscle end cause enhythmlas. In order to study whether nonunIform extdetiincontraction coupling (ECC) ceueee TPCe and Ca* waves, we prepared 11 ret trebsculse with nonunlfam ECC created by local exposure to a smell jet of sallne solution Including 5mM caffeine (CAF) or 2CmM P.S-butanedkne monoxime (BDM). me jet wee applied perpendicularly to the muscle from a glass pipMe (-1 OOpm tip) at a constant flow (O.OSml!min). Force was measured with a etlkon strain gauge. sarcomsre length with laser diffraction techniquas and [Co?, with eleotrophoretiially injected Furs-2 end en image Intenslfied CCD camera. In the region where the trabecula wee exposed to CAF or BDM (-6Wpm). sarcomerss reversibly etrekhed during the twitch, while outside the jet solutkn they continued to shorten, Indketlng locally decreased ECC. Car wavse could be lnducsd, in a controlled manner, from the border between shortening and stretched rsgions following 2.5Hz stimulus trains (75sec, 23-C. [Ce’\=Z.OmM). Ce” waves invariably started late during relaxation of the last stimulated twitch, corresponding to 814% (CAF; n=14) and SS&% (BDM; n=S) relaxation of active fcme. and then propegeted toward the normal regions at velocbies of 0.75+.0.10 (CM) end 0.B540.14mm/eec Arrhythmias also could be induced using this method. These rssulte show that nonuniform ECC, lrraepalve of local damage, can cause Ca” waves end arrhythmlee and that Ca* dksodated from myofllaments during relexatkn in the border xons may play en Important role in the Initiation c Cab waves I