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.

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.

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 Ca(2+)/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 ß-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 beta-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 Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) 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 beta-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.