Working Heart Perfusion Research Articles

Update on Lung Transplantation using Donation after Cardiac Death in NSW

P< 0.05) with hydraulic permeability and burst pressure equal to autologous conduits (HP: IMA 0ml/min/cm2, EC 0 P=ns; BP: IMA 2267 mmHg, EC 1922 P=ns). Fluorescence microscopy confirmed endothelial proliferation on electrospun fibres, and thrombogenicity studies demonstrated reducedplatelet attachment on rHE scaffolds. Discussion: Synthetic coronary conduits constructed using rHE/PCL fibres match the mechanical properties of the IMA. In addition, these scaffolds exhibit reduced thrombogenicity and the ability to support endothelialisation. The elastic conduits will be implanted as carotid artery interposition grafts in rabbits for assessment in vivo before ultimate study as coronary bypass graft candidates. These novel conduits show great potential as a synthetic internal mammary artery, the “Holy Grail” of coronary revascularisation surgery. doi:10.1016/j.hlc.2010.10.034 Update on Lung Transplantation using Donation after Cardiac Death in NSW E. Granger ∗, P. Spratt, M. Harkess, P. Jansz, K. Dhital, P. Macdonald, M. Malouf, A. Glanville The Lung TransplantUnit, St. Vincent’s Hospital, Darlinghurst, ever it will probably augment transplant numbers by only 15%. Current results are excellent despite “warm ischaemia”. Donors with longer warm ischaemic times and more “marginal” gas exchange may now be considered. doi:10.1016/j.hlc.2010.10.035 Beating Heart Preservation of Donor Hearts for Transplantation—A Working Heart Perfusion System for Pig Hearts to Extend Organ Preservation Time P. Pritzwald-Stegmann1,∗, J. Passage1,2, T. Muller 2, A. Muhle2, S. Krabbes1,2, S. Dhein2, F.-W. Mohr2 1Monash Medical Centre, Clayton, Australia Q4 2 Heart Centre Leipzig, Leipzig, Germany Introduction:Donor heart ischaemic time places significant geographic constraints on organ retrieval. Constant pressure perfusion systems can double current time limits in a pig model of heart transplantation from 4 to 8h, but the yield of successfully preserved hearts is low. The aim of this studywas to develop aworking heart perfusion system that would extend donor heart preservation time and allow the subsequent assessment of organ function post reperfusion. Methods: Beating hearts were harvested from 61± 3 kg pigs (n= 6) andperfused via a double lumen cannula using Sydney, NSW, Australia Introduction: Donation after cardiac death (DCD) from Maastrict category 3 donors consists of approximately 12% of lung transplant activity in New South Wales. We review the results of DCD lung transplants and discuss the future direction of the program at St. Vincent’s Hospital. Methodology: Since commencing in November 2007 there have been 25 referrals for DCD, leading to 11 attempted retrievals. Only 9 retrievals resulted in lung donation. Elective withdrawal of life-sustaining support and certification of death (limit of 60min fromwithdrawal) occurred in the Intensive Care Unit or Operating Theatre. Results: Average recipient age was 35 years (range 15–61) and aetiology of respiratory failure included cystic fibrosis (2), bronchiectasis (1), bronchiolitis obliterans (1), emphysema (3), interstitial lung disease (1) and pulmonary hypertension (1). The donor group average age was 37 years (range 14–56) and average PaO2/FiO2 ratio 412 (±51). Causes of death included subarachnoid haemorrhage (2), Traumatic head injury (2), hypoxic brain injury (3) and embolic CVA (2). Mean time to asystole after withdrawal of treatment was 27min (range 9–57), mean warm ischaemic time 19min (range 10–31) and mean cold ischaemic time (left lung) 279min (range 241–320). Early gas exchange was excellent: an average PaO2/FiO2 of 324 (141–503). Average intensive care stay was 5 days (3–10) and length of stay 23 days (12–36). All patients are still alive. Conclusion: Organ donation remains at a challenging low level in Australia. Donation after cardiac death may increase the “donor organ pool”, howaLangendorff-basedperfusioncircuit.Workingheart conditionswere created, at physiological perfusion pressures. After 7 hours, the hearts were arrested with cold HTK cardioplegia solution and underwent 1 hour of warm ischaemia. The hearts were subsequently reperfused in a non-working state at constant pressure for 1 hour and then weaned to again perform under the original working heart conditions. Pmax, max dp/dt, min dp/dt were monitored throughout. Tissue adenosine triphosphate (ATP) and malondialdehyde (MDA) were measured to assess myocardial energy content and oxidative stress respectively. Induction of apoptosis was assessed by staining for AIF. Results: All 6 hearts were successfully harvested, preserved and weaned following reperfusion. Pmax achieved during the working heart mode was higher than in the donor animal (142 vs 86mmHg, p= 0.002). There was no significant overall deterioration in systolic (max dp/dt 2030 vs 1812mmHg/s, p= 0.521) or diastolic (min dp/dt 1200 vs 1225mmHg/s, p= 0.902) function throughout preservation. ATP stores recovered to pre harvest levels (23.6 vs 22.1 pmol/ g, p= 0.57) and oxidative stress was well controlled (1.6 vs 1.8 mol/g, p= 0.926). Apoptosis levels were comparable to hearts that underwent conventional 4 h hypothermic arrest prior to transplantation (15.7 vs 12.7/hpf, p= 0.741). Discussion: A working heart perfusion system can be utilised to successfully increase donor heart preservation times. The system allows the real-time monitoring of donor organ function and canmimic the ischaemia and reperfusion events that occur during transplantation. It permits the specific modification of preservation parame-

High dietary fat promotes a normal metabolic phenotype in the infarcted rat heart

Lipid accumulation is suspected to induce &amp; exacerbate cardiac dysfunction. However, work in our lab has shown high dietary fat (SAT) subsequent to myocardial infarction results in less dysfunction as compared to a standard diet despite increased tissue lipids. This is accompanied by improved mitochondrial function suggesting SAT protection involves energy metabolism. This study sought to evaluate whether SAT alters the energy substrate utilization profile after cardiac infarction. Coronary ligated (HF) or sham (SH) rats were fed SAT or normal chow (NC) for 8 weeks. Working heart perfusions were performed to evaluate function (output, heart rate, &amp; oxygen uptake) &amp; metabolism (glucose &amp; fatty acid oxidation) at low &amp; high workloads. At low workload, cardiac output &amp; oxygen uptake were decreased &amp; glucose oxidation was increased in HF‐NC, but not in HF‐SAT. High workload data suggest a similar trend, but the protective effects were diminished. Lower oxygen use &amp; increased glucose reliance implies a switch from fatty acids in the HF‐NC hearts, a hallmark of heart failure. However, the lack of these differences in the HF‐SAT at low workload suggests SAT does indeed modulate energy metabolism of the infarcted heart. This work supports the hypothesis that high dietary fat after infarction promotes a normal metabolic phenotype &amp; is part of the mechanism by which high fat is protective of cardiac function.

Tolerance of the developing cyanotic heart to ischemia-reperfusion injury in the rat

Whether chronic hypoxia attenuates myocardial ischemia-reperfusion injury remains controversial because conflicting data have been reported probably due to the existence of many factors influencing the functional recovery of hearts. These factors include the differences of species, the time at which hypoxia begins, the degree of hypoxia, and so on. Regarding chronic hypoxia from birth, so far the only available data are based on findings in rabbit hearts. The purpose of this study was to describe the effect of chronic hypoxia from birth on myocardial reperfusion injury in the rat heart. Normoxic hearts were obtained from rats housed in ambient air for 6 weeks (normoxic group); hypoxic hearts were obtained from rats housed in a hypoxic chamber (13%-14% oxygen) from birth for 6 weeks (hypoxic group). Isolated, crystalloid perfused working hearts were subjected to 30 min of global normothermic ischemia followed by 15 min of reperfusion; functional recovery was then measured in the two groups. The excretion of cyclic guanosine monophosphate (cGMP) in the coronary drainage was measured at the end of the preischemia and reperfusion periods. The percent recovery of the left ventricular developed pressure and the first derivative of left ventricular pressure were significantly better in the hypoxic group than in the normoxic group. cGMP excretion in the coronary drainage was significantly increased during both the preischemia and reperfusion periods. Chronic hypoxia from birth increased myocardial tolerance to ischemia-reperfusion injury with increased cGMP synthesis in the isolated heart model in rats.

Impaired contractile function and mitochondrial respiratory capacity in response to oxygen deprivation in a rat model of pre‐diabetes

Obesity is a major contributor to the global burden of disease and is closely associated with the development of type 2 diabetes and cardiovascular diseases. This study tested the hypothesis that mitochondrial respiratory capacity of the pre-diabetic heart is decreased leading to impaired contractile function and tolerance to ischaemia/reperfusion. Eight-week-old male Wistar rats were fed a high caloric diet for 16 weeks after which anthropometric, metabolic, cardiac and mitochondrial parameters were evaluated vs. age-matched lean controls. Cardiac function (working heart perfusions) and mitochondrial respiratory capacity were assessed at baseline and in response to acute oxygen deprivation. Rats fed the high caloric diet exhibited increased body weight and visceral fat vs. the control group. Heart weights of obese rats were also increased. Triglyceride, fasting plasma insulin and free fatty acid levels were elevated, while high-density lipoprotein cholesterol levels were reduced in the obese group. Contractile function was attenuated at baseline and further decreased after subjecting hearts to ischaemia-reperfusion. Myocardial infarct sizes were increased while ADP phosphorylation rates were diminished in obese rats. However, no differences were found for mtDNA levels and the degree of oxidative stress-induced damage. These data show that decreased mitochondrial bioenergetic capacity in pre-diabetic rat hearts may impair respiratory capacity and reduce basal contractile function and tolerance to acute oxygen deprivation.

Abstract 5381: Insulin-Stimulated Glucose Oxidation is Increased in Hearts from High Fat Diet-Induced Obese Mice Lacking Malonyl CoA Decarboxylase

OBJECTIVE - Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial fatty acid metabolites that have been proposed to cause myocardial insulin resistance and dysfunction. Our goal was to determine the effect of DIO on myocardial fatty acid metabolite accumulation and how this is altered when mitochondrial fatty acid uptake is inhibited. This was achieved by using mice lacking malonyl CoA decarboxylase (MCD−/−), which have higher levels of malonyl CoA, an endogenous inhibitor of mitochondrial fatty acid uptake. METHODS - Wild type (WT) and MCD−/− mice were fed a low (4% kcal from lard) or high (60% kcal from lard) fat diet for 12 weeks to determine the effect of DIO on the intra-myocardial accumulation of long chain acylcarnitines, long chain acyl CoAs, triglycerides (TGs), and ceramides. A parallel feeding study was performed to assess myocardial function and energy metabolism in isolated working hearts in the absence/presence of insulin. RESULTS - We demonstrate that MCD−/− mice do not accumulate intramyocardial long chain acylcarnitines to the same extent as WT mice following DIO (0.56 ± 0.10 vs. 0.28 ± 0.07 pmol myristoylcarnitine/mg protein, P &lt;0.05), but do accumulate similar amounts of long chain acyl CoAs (3.88 ± 0.34 vs. 4.35 ± 1.19 nmol/g wet weight). Interestingly, DIO only lead to an accumulation of TGs in the hearts of MCD−/− mice (3.29 ± 0.62 vs. 10.92 ± 3.72 μmol/g wet weight, P &lt;0.05). Despite this elevation in TGs, MCD−/− mice showed increased insulin-stimulated glucose oxidation (2.46 ± 0.25 vs. 1.74 ± 0.18 fold increase, P &lt;0.05) during aerobic isolated working heart perfusions and did not elicit any dysfunction. CONCLUSIONS - Our data reveal discordance between myocardial TG accumulation and glucose metabolism, suggesting that TG buffers against toxic lipids, and that inhibition of mitochondrial fatty acid oxidation does not cause myocardial dysfunction following DIO.

Cardiac-restricted Expression of the Carboxyl-terminal Fragment of GRK3 Uncovers Distinct Functions of GRK3 in Regulation of Cardiac Contractility and Growth: GRK3 CONTROLS CARDIAC α1-ADRENERGIC RECEPTOR RESPONSIVENESS

G protein-coupled receptor kinase-2 and -3 (GRK2 and GRK3) in cardiac myocytes catalyze phosphorylation and desensitization of different G protein-coupled receptors through specificity controlled by their carboxyl-terminal pleckstrin homology domain. Although GRK2 has been extensively investigated, the function of cardiac GRK3 remains unknown. Thus, in this study cardiac function of GRK3 was investigated in transgenic (Tg) mice with cardiac-restricted expression of a competitive inhibitor of GRK3, i.e. the carboxyl-terminal plasma membrane targeting domain of GRK3 (GRK3ct). Cardiac myocytes from Tg-GRK3ct mice displayed significantly enhanced agonist-stimulated alpha(1)-adrenergic receptor-mediated activation of ERK1/2 versus cardiac myocytes from nontransgenic littermate control (NLC) mice consistent with inhibition of GRK3. Tg-GRK3ct mice did not display alterations of cardiac mass or left ventricular dimensions compared with NLC mice. Tail-cuff plethysmography of 3- and 9-month-old mice revealed elevated systolic blood pressure in Tg-GRK3ct mice versus control mice (3-month-old mice, 136.8 +/- 3.6 versus 118.3 +/- 4.7 mm Hg, p < 0.001), an observation confirmed by radiotelemetric recording of blood pressure of conscious, unrestrained mice. Simultaneous recording of left ventricular pressure and volume in vivo by miniaturized conductance micromanometry revealed increased systolic performance with significantly higher stroke volume and stroke work in Tg-GRK3ct mice than in NLC mice. This phenotype was corroborated in electrically paced ex vivo perfused working hearts. However, analysis of left ventricular function ex vivo as a function of increasing filling pressure disclosed significantly reduced (dP/dt)(min) and prolonged time constant of relaxation (tau) in Tg-GRK3ct hearts at elevated supraphysiological filling pressure compared with control hearts. Thus, inhibition of GRK3 apparently reduces tolerance to elevation of preload. In conclusion, inhibition of cardiac GRK3 causes hypertension because of hyperkinetic myocardium and increased cardiac output relying at least partially on cardiac myocyte alpha(1)-adrenergic receptor hyper-responsiveness. The reduced tolerance to elevation of preload may cause impaired ability to withstand pathophysiological mechanisms of heart failure.

Myocardial functional and metabolic parameters in heart failure following chronic high fat feeding

Heart failure (HF) is a complex process that remains to be fully understood, but involves multiple factors. Excess dietary fat is a risk factor for cardiovascular disease, but the role of fatty acids in HF progression has yet to be defined. Lipid accumulation in non-adipose tissue is known to cause a number of pathological events implicating fatty acids in cardiac pathology progression. However, the relationship between dietary lipid and contractile dysfunction is complicated; recent data from human and animal studies suggest dietary fat may be protective following infarction. Previous work in our laboratory using coronary artery ligation demonstrates that rats fed high fat after cardiac injury results in less contractile dysfunction and increased mitochondrial respiration despite increased tissue triglyceride. These results suggest an alteration in substrate utilization that benefits the injured heart. The current study aimed to characterize the impact of high fat on cardiac performance and substrate utilization in rats using a working heart perfusion model. Cardiac output, oxygen consumption (MVO2), and glucose oxidation were measured. Under normal conditions, HF animals had decreased MVO2 with no change in glucose oxidation. However, high fat feeding resulted in increased MVO2 with lower glucose oxidation in sham (SH) and HF animals. Under conditions of increased workload, HF animals had lower cardiac output, MVO2, and glucose oxidation. Furthermore, both SH and HF animals fed high fat had lower cardiac output and glucose oxidation than their respective controls fed normal chow. These results suggest that high dietary fat may not alter cardiac function under normal conditions, but may be deleterious for contractile and metabolic function during increased physiological stress.

Short-term effects of pressure overload on the expression of genes involved in calcium homeostasis

We investigated whether in the isolated perfused rat heart acute pressure overload may affect the expression of genes involved in calcium homeostasis, namely sarcolemmal L-type Ca2+ channel, Na+/Ca2+ exchanger, sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and ryanodine receptor. Hearts were subjected to 210 min of perfusion under the following conditions: (i) standard working heart perfusion with preload and afterload set at 20 and 100 cm, respectively; (ii) working heart perfusion at high afterload (180 cm); (iii) retrograde infusion of St. Thomas' Hospital cardioplegic solution. In all models gene expression was determined by RT-PCR. Significant decrease in the expression of the sarcoplasmic reticulum Ca2+-ATPase gene was observed in the high afterload group. No significant change in the expression of any other gene was observed in any group. The reported effect was not detected after 60 min of perfusion, and it was blunted in the presence of the protein kinase C inhibitor chelerythrine, while the calcineurin inhibitor cyclosporin A was ineffective. In conclusion, the sarcoplasmic reticulum Ca2+-ATPase gene is downregulated after short-term (210 min) perfusion at high afterload, possibly through a protein kinase C-dependent pathway. This mechanism might play a relevant pathophysiological role in the response to pressure overload and in the development of hypertrophy.

Criteria for the multiple use of isolated perfused hearts in electrophysiological and metabolic experiments / Kriterien für die Mehrfachnutzung von isoliert perfundierten Schweineherzen für elektrophysiologische und metabolische Untersuchungen

Both ethical and economic restrictions limit the availability of porcine hearts for in vitro perfusion experiments. Therefore, we tested the feasibility of multiple use of in vitro perfused working hearts for electrophysiological and metabolic investigations. Pig hearts (n=7) rejected for originally planned haemodynamic measurements because of exclusion criteria were perfused in a four-chamber working heart mode. All hearts were kept in steady-state conditions on a low haemodynamic level over 2 h during 75-channel ECG recordings and NADH fluorescence measurements before and after norepinephrine (NE) was administered. QRS and QT interval durations were in a range comparable to in vitro studies and, like QRS and T amplitudes, were found to be sensitive markers of the changing condition of the isolated heart preparation, as myocardial oedema leads to prolonged QRS and QT intervals and declining ECG voltage amplitudes. A change in NADH fluorescence following NE administration was observed in the first 150 min of perfusion, but not later. Considering a time frame of 120 min, multiple use of isolated perfused porcine hearts with low-level haemodynamics may allow a broad spectrum of investigations and could therefore represent a possibility of overcoming the restricted availability of porcine hearts.

A Conserved Role for Phosphatidylinositol 3-Kinase but Not Akt Signaling in Mitochondrial Adaptations that Accompany Physiological Cardiac Hypertrophy

Physiological cardiac hypertrophy is associated with mitochondrial adaptations that are characterized by activation of PGC-1alpha and increased fatty acid oxidative (FAO) capacity. It is widely accepted that phosphatidylinositol 3-kinase (PI3K) signaling to Akt1 is required for physiological cardiac growth. However, the signaling pathways that coordinate physiological hypertrophy and metabolic remodeling are incompletely understood. We show here that activation of PI3K is sufficient to increase myocardial FAO capacity and that inhibition of PI3K signaling prevents mitochondrial adaptations in response to physiological hypertrophic stimuli despite increased expression of PGC-1alpha. We also show that activation of the downstream kinase Akt is not required for the mitochondrial adaptations that are secondary to PI3K activation. Thus, in physiological cardiac growth, PI3K is an integrator of cellular growth and metabolic remodeling. Although PI3K signaling to Akt1 is required for cellular growth, Akt-independent pathways mediate the accompanying mitochondrial adaptations.

Dietary red palm oil reduces ischaemia–reperfusion injury in rats fed a hypercholesterolaemic diet

We have previously shown that dietary red palm oil (RPO) supplementation improves functional recovery in hearts subjected to ischaemia-reperfusion. However, little knowledge exists concerning the effects of RPO supplementation of a high-cholesterol diet on ischaemia-reperfusion injury. The signalling mechanisms responsible for RPO's effects in the presence of cholesterol also remain to be elucidated. Therefore, the aim of the present study was to examine the effects of RPO, given with a high-cholesterol diet, on mitogen-activated protein kinase (MAPK) phosphorylation and apoptosis. Long-Evans rats were fed a control diet, a control diet containing 2% cholesterol, or a control diet containing 2% cholesterol and 7 g RPO per kg (CRPO) for 5 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured after which hearts were freeze-clamped and used to assess MAPK phosphorylation and to evaluate apoptosis. Cholesterol supplementation caused a poor aortic output (AO) recovery compared with the control group (35.5 (sem 6.2) v. 55.4 (sem 2.5) %), but when RPO was added, the percentage AO increased significantly. The cholesterol group's poor AO was associated with a significant increase in p38-MAPK phosphorylation, whereas the CRPO-supplemented group showed as significant reduction in p38-MAPK phosphorylation when compared with the cholesterol-supplemented group. This significant reduction in p38-MAPK was also associated with reduced apoptosis as indicated by significant reductions in caspase-3 and poly(ADP-ribose) polymerase cleavage.

Dramatic Accumulation of Triglycerides and Precipitation of Cardiac Hemodynamic Dysfunction during Brief Caloric Restriction in Transgenic Myocardium Expressing Human Calcium-independent Phospholipase A2γ

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Overexpression of angiotensinogen in the myocardium induces downregulation of the fatty acid oxidation pathway

Heart failure is associated with downregulation of the fatty acid oxidation pathway in the ventricular myocardium. Since angiotensin II plays a critical role in myocardial phenotypic changes associated with heart failure, we investigated the effect of chronic angiotensin II stimulation on the fatty acid oxidation pathway in transgenic (TG) mice with targeted overexpression of angiotensinogen in the myocardium (TG1306/1R mice). TG1306/R1 mice progressively developed left ventricular hypertrophy. After 12 months, approximately half of the mice exhibited signs of heart failure including increased lung weight index [> + 2 SD of age-matched wild-type (WT) mice] and 5-fold increase of myocardial brain natriuretic peptide expression. Myocardial mRNA and protein expression of peroxisome proliferator-activated receptor α (PPARα) progressively decreased in both WT and TG1306/R1 mice during the 12 months observation period, but much more pronounced in TG1306/R1 mice. Concomitantly, mRNA expression of enzymes of fatty acid oxidation (medium-chain acyl CoA dehydrogenase, MCAD; carnitine palmitoyl transferase I, CPT-I) was reduced in TG1306/R1 compared with age-matched WT mice. However, protein expression of MCAD and CPT-I was decreased concomitantly only in TG mice with criteria of heart failure. Correspondingly, myocardial oxidation of palmitate, measured during ex vivo working heart perfusion, was reduced by 25% in TG1306/R1 mice with heart failure. These results demonstrate that angiotensin II-induced cardiac hypertrophy is associated with reduction of PPARα and of mRNA expression of enzymes of fatty acid metabolism relative to age-matched WT mice. However, both protein expression of fatty acid oxidation enzymes and the rate of fatty acid oxidation remain unchanged unless heart failure occurs, suggesting the involvement of posttranscriptional mechanisms in the metabolic changes associated with heart failure.

Exercise‐Induced Increase in Pro‐ and Macroglycogen Content in Hearts from Type 1 Diabetic Mice

Diabetes adversely affects glucose metabolism, resulting in elevated levels of heart glycogen. Exercise training increases glycogen synthesis activity in diabetes; therefore the aim of this study was to examine the effects of exercise on glycogen content in hearts from streptozotocin-induced (insulin-deficient for duration of study) type 1 diabetic mice (T1D) mice compared to CD-1 controls. Mice were exercised by running for 60 min/d, 5 d/wk for 6 wk at low-moderate intensity. Hearts from T1D and control mice at 12 wk of age were subjected to a 60 min working heart perfusion with glucose and palmitate, prior to glycogen measurements (μmol/g dw) as total glycogen (Gt), proglycogen (PG) and macroglycogen (MG). Gt for T1D sedentary (SED) hearts was not significantly different from SED or exercised (EX) non-diabetic, CD1 controls. In T1D SED hearts, PG contributed 60% of total glycogen, which was significantly elevated compared to EX controls but not to SED controls. EX T1D hearts showed a nearly 2-fold increase in Gt compared to SED diabetic hearts (935±86 vs 584±20) with PG and MG contributing 558±21 and 436±80 glucosyl units respectively. There are intriguing aspects of this data: i) there was no difference in Gt between SED T1D and controls implying glycogen synthesis is not impaired in T1D hearts. (ii) Elevated PG levels in T1D hearts reflects a distinct selective process in glycogen formation compared to controls, suggesting T1D hearts form new granules at the expense of MG to maintain Gt. (iii) An exercise-induced improvement in glucose disposal may account for elevated PG and MG levels in EX T1D perfused hearts. Supported by CIHR (DS) and Genome Alberta (JS).

Exercise does not Attenuate Increased Fatty Acid Metabolism in Ex Vivo Diabetic Hearts

Diabetes mellitus produces an altered metabolic phenotype in the heart, with increased fatty acid oxidation and decreased glucose metabolism. This study determined whether regular exercise training could attenuate the consequences of enhanced cardiac fatty acid (FA) oxidation in streptozotocin-injected type 1 diabetic mice (T1D). Control (CD-1) and diabetic mice were exercised (EX) by running for 60 min/d, 5 d/wk for 6 wk at low-moderate intensity. Non-exercised sedentary (SED) groups were also studied. Hearts from T1D and control mice at 12 weeks of age were subjected to a 60-min working heart perfusion with glucose and palmitate. Oxidation of radiolabeled palmitate (complexed to albumin) was measured (μmol/min/g dry weight) along with parameters of contractile function. Diabetic mice exhibited hyperglycemia compared to CD-1 controls (P<0.01). Importantly, EX T1D group showed a significant 12% reduction in plasma glucose levels compared to T1D SED mice (41.4 vs 50.3 mM). Hearts from CD-1 and T1D groups showed no changes with exercise for any of the measured parameters of contractile function. Palmitate oxidation was increased by nearly 2-fold in T1D SED hearts compared to CD-1 SED hearts (0.72±0.12 vs 0.40±0.05 μmol/min/g dry weight). However, steady-state rates of palmitate oxidation were not significantly different between T1D SED and T1D EX hearts (0.72±0.12 vs 0.63±0.08). These results suggest fatty acid oxidation rates during perfusion, although significantly elevated in the diabetic heart, are not altered by exercise. As well 6 weeks of low-moderate exercise is not enough to improve diabetic cardiac function. However, lower blood glucose levels in T1D EX mice suggest a beneficial effect of exercise on whole-body glucose utilization. Supported by CIHR (DS).

A Newly Synthesised Molybdenum/Ascorbic Acid Complex Alleviates Some Effects of Cardiomyopathy in Streptozocin-Induced Diabetic Rats

Exogenous insulin does not prevent cardiac failure in patients with type 1 diabetes mellitus and a cardioprotective insulin mimic is greatly needed. Certain transition metals are known to act as insulin mimics and may be cardio- protective. In this study, the ability of a newly synthesised molybdenum/ascorbic acid complex to strengthen cardiac function was investigated. Male CD rats were assigned to one of five groups: non-diabetic control, non-diabetic control treated with molybdenum/ascorbic acid complex, diabetic treated with sodium ascorbate, diabetic treated with molybdenum/ascorbic acid complex and untreated diabetics. Type 1 diabetes was induced by streptozocin injection. Once diabetes was confirmed, treatment was initiated by adding either the molybdenum/ascorbic acid complex or sodium ascorbate to the drinking water and continued for 6 weeks. Following the treatment period, the animals were terminated, and their hearts were excised and mounted in a working heart perfusion apparatus. Blood samples were taken for plasma glucose and plasma lipid level determination. Cardiac function was evaluated using 1 hour of low-flow ischaemic stress followed by 30 minutes of reperfusion. Hearts from the animals treated with the molybdenum/ascorbic acid complex displayed the best aerobic performance of all the diabetic animals. Blood glucose levels and blood lipid levels were significantly lower in animals treated with the complex than in other diabetic animals. The group treated with the complex also had a lower drinking rate than the other diabetic groups. Furthermore, hearts from animals treated with the molybdenum/ascorbic acid complex showed a greater degree of recovery from low-flow ischaemia than any other group. The molybdenum/ascorbic acid complex showed some significant insulin-mimic and cardioprotective effects. Further development of this complex could provide a drug useful for alleviating some of the cardiovascular problems associated with diabetes mellitus.

Supplementation of Nucleoside-Nucleotide Mixture Enhances Functional Recovery and Energy Metabolism Following Long-Time Hypothermic Heart Preservation

OG-VI, a well-balanced mixture of nucleoside and nucleotides, has been demonstrated to have a favorable effect on energy metabolism. In this study, we tested the hypothesis that addition of OG-VI to the University of Wisconsin solution can improve the cardiac functional recovery following long-time hypothermic preservation. Forty-two male Wistar rats were randomized into four groups. After 30-min of isolated working heart perfusion, the rat hearts were arrested with St. Thomas cardioplegic solution and preserved at 4 degrees C in saline, OG-VI, UW, and UW+OG-VI, respectively. After 12-h of preservation, the hearts were reperfused for 60-min during which the recovery of cardiac functions were monitored continuously. Myocardial adenine nucleotides were analyzed using high-performance liquid chromomatograph. In the UW+OG-VI group, the recovery of cardiac output, coronary flow, aortic flow, rate-pressure product, left ventricle stroke volume, and stroke work were significantly higher than other groups (P < 0.05). Furthermore, all phosphate high-energy compounds were significantly higher in the UW+OG-VI group than in the other groups (P < 0.05). Coronary vascular resistance and myocardial wet/dry weight ratio were obviously lower in the UW+OG-VI group, compared to the other groups (P < 0.05). Heart function was better recovered when nucleoside-nucleotide mixture was added to UW solution during long-time hypothermic rat heart preservation. The mechanism is not totally clear, but enhancement of high-energy phosphate production is possible.

P38-MAPK and PKB/Akt, possible role players in red palm oil-induced protection of the isolated perfused rat heart?

It has been shown that dietary red palm oil (RPO) supplementation improves reperfusion function. However, no exact protective cellular mechanisms have been established. To determine a potential mechanism for functional improvement, we examined the regulation of both mitogen-activated protein kinases (MAPKs) and PKB/Akt in the presence and absence of dietary RPO supplementation in ischemia/reperfusion-induced injury. Wistar rats were fed a control diet or control diet plus 7 g RPO/kg diet for 6 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 min of total global ischemia. Hearts subjected to the same conditions were freeze clamped and used to characterize the degree of phosphorylation of extracellular signal-regulated kinase, p38, c-Jun NH 2-terminal protein kinase (JNK) and PKB/Akt. Dietary RPO supplementation significantly improved aortic output recovery (72.1±3.2% vs. 54.0±3.2%, P<.05). This improved aortic output recovery was associated with significant increases in p38 and PKB/Akt phosphorylation during reperfusion when compared with control hearts. Furthermore, a significant decrease in JNK phosphorylation and attenuation of poly(ADP-ribose) polymerase cleavage occurred in the RPO-supplemented group during reperfusion. Our results suggest that dietary RPO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. These changes in phosphorylation were associated with improved functional recovery and reduced cleavage of an apoptotic marker, arguing that dietary RPO supplementation may confer protection via the MAPK and PKB/Akt signaling pathways during ischemia/reperfusion-induced injury.

Combining the hDAF transgene with the GP IIb/IIIa inhibitor tirofiban improves heart performance and reduces myocardial damage following hyperacute rejection in an ex vivo perfusion model

Xenograft rejection is associated with vascular injury resulting at least in part from platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis. Assuming that complement activation is a major determinant of humoral immune reactions bringing about platelet-endothelial cell interactions, we tested the effects of the specific platelet glycoprotein IIb/IIIa inhibitor tirofiban in combination with the human decay accelerating factor (hDAF) transgene on hyperacute rejection of pig hearts. Four groups were studied in a working heart–perfusion model. Pig hearts transgenic for hDAF and nontransgenic pig hearts were perfused with human blood containing tirofiban or with unmodified human blood. Cardiac output, stroke work index, and creatine phosphokinases were measured for the evaluation of the extent of myocardial damage. Consumption of complement components was determined. Endothelial deposition of fibrin and intravascular thrombosis were evaluated. Tirofiban improved cardiac output and stroke work index of nontransgenic pig hearts and was able to further increase hemodynamic function of hDAF transgenic pig hearts. Low levels of creatine phosphokinases also revealed a cardioprotective effect of tirofiban. However, a further extension of the survival of hDAF transgenic pig hearts could not be achieved, although tirofiban prolonged beating time of nontransgenic pig hearts. Tirofiban was able to reduce the consumption of complement components independently of hDAF. Intravascular evidence of fibrin and thrombosis tended to be particularly reduced by the combination of tirofiban and hDAF. Thus, the application of tirofiban together with hDAF improves the performance of pig hearts by reducing myocardial damage and intravascular thrombosis.

Age-dependent changes in metabolism, contractile function, and ischemic sensitivity in hearts from db/db mice.

Glucose and palmitate metabolism and contractile function were measured with ex vivo perfused working hearts from control (db/+) and diabetic (db/db) female mice at 6, 10-12, and 16-18 weeks of age. Palmitate oxidation was increased by 2.2-fold in 6-week-old db/db hearts and remained elevated in 10- to 12- and 16- to 18-week-old hearts. Carbohydrate oxidation was normal at 6 weeks but was reduced to 27 and 23% of control at 10-12 and 16-18 weeks, respectively. At 6 weeks, db/db hearts exhibited a slight reduction in mechanical function, whereas marked signs of dysfunction were evident at 10-12 and 16-18 weeks. Mechanical function after ischemia-reperfusion was examined in hearts from male mice; at 6 weeks, db/db hearts showed normal recovery, whereas at 12 weeks it was markedly reduced. Fatty acid oxidation was the predominant substrate used after reperfusion. Thus, diabetic db/db hearts exhibit signs of a progressive cardiomyopathy; increased fatty acid oxidation preceded reductions in carbohydrate oxidation. Postischemic recovery of function was reduced in db/db hearts, in parallel with age-dependent changes in normoxic contractile performance. Finally, peroxisome proliferator-activated receptor-alpha treatment (3 weeks) did not affect sensitivity to ischemia-reperfusion, even though carbohydrate oxidation was increased and palmitate oxidation was decreased.