Zeinab Hegab

Role of advanced glycation end products in cardiovascular disease

Advanced glycation end products (AGEs) are produced through the non enzymatic glycation and oxidation of proteins, lipids and nucleic acids. Enhanced formation of AGEs occurs particularly in conditions associated with hyperglycaemia such as diabetes mellitus (DM). AGEs are believed to have a key role in the development and progression of cardiovascular disease in patients with DM through the modification of the structure, function and mechanical properties of tissues through crosslinking intracellular as well as extracellular matrix proteins and through modulating cellular processes through binding to cell surface receptors [receptor for AGEs (RAGE)]. A number of studies have shown a correlation between serum AGE levels and the development and severity of heart failure (HF). Moreover, some studies have suggested that therapies targeted against AGEs may have therapeutic potential in patients with HF. The purpose of this review is to discuss the role of AGEs in cardiovascular disease and in particular in heart failure, focussing on both cellular mechanisms of action as well as highlighting how targeting AGEs may represent a novel therapeutic strategy in the treatment of HF.

Plasma membrane calcium pump (PMCA4)/neuronal nitric oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain

Background: Previously we have shown that PMCA4 interacts with nNOS. Results: In PMCA4−/− mice, plasma membrane-associated nNOS protein was delocalized to the cytosol with no change in total nNOS protein. Conclusion: The current study shows that PMCA4-nNOS complex modulates a spatially confined cyclic nucleotide microdomain at the plasma membrane. Significance: Compartmentalization of the PMCA4-nNOS complex has a major role in regulating cardiac contractility. Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca2+/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4−/−) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4−/−, over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium.

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates β-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser16 phospholamban (PLB) phosphorylation as well as Ser22 and Ser23 cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the β-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Advanced glycation end products reduce the calcium transient in cardiomyocytes by increasing production of reactive oxygen species and nitric oxide

Advanced glycation end products (AGE) are central to the development of cardiovascular complications associated with diabetes mellitus. AGE may alter cellular function through cross‐linking of cellular proteins or by activating the AGE receptor (RAGE). However, the signalling molecules involved during AGE stimulation in cardiomyocytes remain unclear. Here, we investigated the effects of AGE treatment on intracellular calcium homeostasis of isolated cardiomyocytes and studied the activation of signalling molecules involved in this process. Treatment of cardiomyocytes with AGE for 24 h resulted in a dose‐dependent reduction in calcium transient amplitude, reaching a maximum 50% reduction at a dose of 1 mg·mL−1. This was accompanied with a 32% reduction in sarcoplasmic reticulum calcium content but without any detectable changes in the expression of major calcium channels. Mechanistically, we observed a significant increase in the production of reactive oxygen species (ROS) in AGE‐treated cardiomyocytes and enhancement of NADPH oxidase activity. This was accompanied with activation of p38 kinase and nuclear translocation of NF‐κB, and subsequently induction of inducible NO synthase (iNOS) expression, leading to excessive nitric oxide production. Overall, our data reveal the molecular signalling that may underlie the alteration of intracellular calcium homeostasis in cardiac myocytes due to AGE stimulation. This may provide new insights into the pathophysiological mechanisms of the development of diabetic cardiomyopathy.

74 Mechanistic Study for the role of advanced glycation end products in the development of diabetic heart failure

Advanced glycation end products (AGEs) are thought to play a crucial role in the development of diabetic complications including heart failure, a leading cause of morbidity and mortality in diabetic patients. However, the molecular mechanisms that underlie the pathophysiological contribution of AGEs to heart failure development are not yet fully understood. We therefore investigated the effects and mechanisms of action of AGEs on isolated neonatal rat cardiomyocytes (NRCM). Standard molecular techniques were applied. Western blot showed that RAGE receptor is expressed in NRCM and adult mouse cardiomyocytes. Incubation of NRCM for 24 h with AGEs showed a dose dependant reduction of calcium transient amplitude with a maximum of 52% at 1 g/l (p<0.01) accompanied with 32% reduction in SR calcium content with no significant changes in the protein expression of calcium handling proteins. We demonstrated a 24% increase (p<0.01) in the production of reactive oxygen species ROS in AGE treated cardiomyocytes mediated through increased NADPH oxidase activity (p<0.05). Subsequent translocation of NF-KB, a transcriptional factor from the cytoplasm to the nucleus together with increased NF-KB activity resulted in a 56% increase in iNOS gene protein expression (p<0.01), a downstream target of NF-KB. The latter was associated with 10% increase in NO production (p<0.05) with subsequent nitrosylation of the Ryanodine receptor shown through immunofluoresence. Changes in calcium transient were completely inhibited when we incubated the cardiomyocytes with inhibitors of NADPH oxidase, NOS or NF-KB prior to their incubation with AGEs. In conclusion, AGEs directly decline cardiomyocytes function through binding to their RAGE receptor leading to calcium handling impairment through increased ROS production inducing activation and translocation of NF-KB to the nucleus. The latter increased transcription of iNOS with increased NO production. Coexistence of ROS and NO favours the production of peroxynitrite that is capable of nitrosylation of key cellular proteins such as the Ryanodine receptor that has a crucial role in cardiac excitation-contraction coupling. This study provides novel insights into the mechanisms of cardiac damage in diabetes that occur independent of vascular disease through AGEs.

012 Differential roles of the plasma membrane calcium pump isoforms 1 and 4 in modulating cardiac contractility

Heart failure is a syndrome currently affecting almost one million people in the UK. Abnormal calcium handling is one the characteristic features of heart failure. The plasma membrane calcium pump (PMCA) is one of the calcium transporters in the cardiomyocytes. Two isoforms of PMCA, PMCA1 and 4, are expressed in the myocardium. Our group has shown previously that PMCA4 modulates the beta-adrenergic response in the heart through its interaction with nNOS. Currently, in order to elucidate the physiological importance of both PMCA isoforms in the modulation of cardiac contractility we generated mice carrying a genetic deletion of either PMCA1 or PMCA4. Given that PMCA4 is a calcium extrusion pump it was unexpected that in vivo basal contractility was enhanced in PMCA4 KO mice (dP/dtmax in 8049±628 vs 6604±296 mmHg/s in KO and WT respectively, p<0.05, n=10). This enhanced contractility was imitated in WT mice by injecting the nNOS specific inhibitor (L-NPA). Ca2+ transients in cardiomyocytes from PMCA4 KO mice showed an increase in amplitude (298.8±24.3 nM calcium vs 492.2±28.2 nM calcium in WT and KO respectively; p<0.05, n=8) with no change in the rate of Ca2+ decay. Again, this phenotype was imitated by nNOS specific inhibition in WT adult CMC. Although, there is no difference in total nNOS protein expression between PMCA4 KO and WT, the nNOS localisation and activity at the sarcolemmal membrane was decreased by 52% in PMCA4 KO. Intracellular cardiac cGMP levels in PMCA4 KO were markedly decreased (619±16.7 fmol/mg protein in KO vs 713.4±24.2 fmol/mg protein in WT) suggesting that the phenotype was likely through nNOS modulation. Since the global deletion of PMCA1 is embryonic lethal, we generated PMCA1 cardiac-specific knockout mice (PMCA1cko) using Cre/LoxP technology. PMCA1cko showed a reduction in the rate of relaxation (logistic τ; 6.9±0.34 vs 5.8±0.29 ms in PMCA1cko and PMCA1flox/flox control respectively; p<0.05, n=11) without any change in cardiac contractility. On the contrary to PMCA4 KO, cardiomyocytes from PMCA1cko revealed a decreased rate of Ca2+ decay (τ, 0.201±0.009 vs 0.164±0.006 ms in PMCA1cko and PMCA1flox/flox respectively; p<0.05, n=16), while Ca2+ transient amplitude remained unchanged. The nNOS protein expression, localisation and activity in PMCA1cko mice were similar to those in PMCA1flox/flox. In conclusion, our results suggest differential rather than redundant roles of the cardiac PMCAs. PMCA4 regulates cardiac signalling through modulation of membrane nNOS activity, while PMCA1 modulates fine tuning of diastolic calcium in the excitation-contraction coupling. The use of non-isoform-specific inhibitors in previous work was unable to detect these differential roles as the effects of PMCA1 and 4 inhibition cancel each other out, at least in part.

008 Spontaneous cardiac hypertrophy and identification of novel hypertrophic pathways in a genetic mouse model that emulates human diabetes

Animal models are key in the exploration of the pathophysiological mechanisms and complications of diabetes mellitus (DM). Most current models have considerable limitations in that they do not faithfully replicate human forms of DM. Recently, a novel human relevant mouse model of diabetes (GENA 348) was identified through the Medical Research Council, Harwell, UK mouse mutagenesis programme in which a point mutation in the glucokinase gene results in severely impaired glucokinase function and significant hyperglycaemia. Similar mutations in the glucokinase gene are known to underlie Maturity Onset Diabetes of the Young Type 2 (MODY 2) in humans. We studied the GENA 348 mouse to determine whether it expresses a cardiac phenotype to provide insight into the pathophysiological mechanisms underlying the development of diabetic cardiomyopathy. Fifteen wild type (WT) controls and eight homozygote mutant (HO) mice had serial echocardiography performed at 3 and 6 months. At 3 months no evidence of cardiac hypertrophy or contractile dysfunction was demonstrated in HO compared to WT mice. By 6 months of age, echo demonstrated development of cardiac hypertrophy in GENA 348 HO mice characterised by a 16% increase in left ventricular mass/body weight (wt 4.28±0.15 vs HO 4.95±0.26, p<0.05), a 25% increase in dPW/dD (WT 23.3±1.0 vs HO 29.1±1.1, p<0.01) and an 18% increase in left ventricular relative wall thickness (WT 0.45±0.01 vs HO 0.53±0.02, p<0.05). No differences in systolic function were observed although significant diastolic dysfunction was also evident with a 31% reduction in the E:A ratio (WT 2.65±0.2 vs HO 1.84±0.19, p<0.05) and a 34% increase in the IVRT (WT 15.2±1.1 ms vs HO 20.5±0.37 ms, p<0.01). Histological staining illustrated cellular hypertrophy with a 60% increase in cardiomyocyte size (WT 238±14 μm2 vs HO 383±26 μm2, p<0.001). Hypertrophic pathways were examined through western blot analysis, a 140% increase in Akt phosphorylation, a key mediator of cardiac hypertrophy and a 65% increase in GSK3β phosphorylation one of its key regulators was observed. We also demonstrate that advanced glycation end products (AGE) were elevated by 86% in the serum of mutant GENA 348 mice. Collectively, these data indicate GENA 348 mutant mice develop a cardiac phenotype including hypertrophy and diastolic dysfunction similar to the clinical manifestations of diabetic cardiomyopathy. Furthermore, a novel hypertrophic pathway has been identified in which AGE stimulation through GSK3β leads to activation of the intracellular Akt pathway. GENA 348 may thus provide a valuable, human-relevant tool for studying the molecular determinants of DM related cardiovascular complications.