Recent reports have implicated the parasympathetic nervous system in the development and progression of heart failure as well as being a potential therapeutic target in heart disease 
. Vagal stimulation can improve outcome in experimental heart failure 
and this can be mimicked by treatment with cholinesterase inhibitors 
. Our previous studies have demonstrated that mice with a systemic decrease in cholinergic tone develop cardiac dysfunction and exhibit many of the characteristics present in cardiac remodeling 
. Importantly, in VAChT KDHOM
mice, these cardiac defects are ameliorated by cholinesterase inhibitor treatment, implicating release of ACh rather than developmentally-induced changes in the control of heart function. Interestingly, in heart failure, transdifferentiation of sympathetic neurons into a cholinergic phenotype has recently been demonstrated, and this appears to have a protective role 
. However, the role of acetylcholine in controlling long-term cardiac function is still poorly understood.
The present study examined whether the decrease in parasympathetic tone in VAChT KDHOM
mice leads to alterations in cardiac gene expression which may contribute to the observed cardiac dysfunction. Our microarray analysis revealed a number of transcriptional changes with a total of 71 genes being significantly different between these mice. Interestingly, transcript and protein levels of two purine nucleoside phosphorylases (Pnp and Pnp2) were significantly increased in the VAChT mutant mice. Both Pnp and Pnp2 are important enzymes responsible for the conversion of inosine to hypoxanthine and have also been shown to metabolize adenosine into adenine, especially under conditions of cardiac stress 
The increased levels of Pnp and Pnp2 in VAChT KDHOM
mice may lead to the increased production of hypoxanthine in mutant hearts 
. Interestingly, endothelial cells in the heart appear to be responsible for the majority of adenosine uptake 
. In addition, they are responsible for the metabolism of adenosine into several compounds, including hypoxanthine 
. Increased levels of this adenosine metabolite may serve a key role in the cardiac dysfunction observed in mutant mice. For example, hypoxanthine, produced in endothelial cells, can be taken up by the ENT and ENBT1 transporters into myocytes 
, where it can be further metabolized into xanthine and urate 
. These metabolites contribute to the production of ROS 
, increased levels of which have been shown to play a role in cardiac and vascular dysfunction in both ischemic and non-ischemic cardiomyopathies 
. Interestingly, we observed an increase in the levels of ROS in ventricular cardiomyocytes isolated from VAChT KDHOM
mice. Oxygen free radicals contribute to declining cardiac function during heart failure via many different mechanisms and result in damage to the myocardium 
. They can also have detrimental effects specifically in cardiomyocytes as they can activate cell death through both necrotic and apoptotic pathways 
. Future studies will be necessary to further characterize the mechanisms which lead to increased ROS production in the mutant mice as well as determine the physiological importance of these oxygen free radicals and their role in the observed cardiac dysfunction. However, it is tempting to speculate that these alterations may play a role in the dysfunction found in VAChT mutant mice.
It is important to note that ADP levels are upregulated in heart failure suggesting that failing cardiac tissue utilizes greater amounts of ATP 
. Under normal conditions, the vasodilatory actions of adenosine may be able to compensate for this increased utilization of ATP by myocardial tissue. However, VAChT KDHOM
mice show increased levels of Pnp which has previously been shown to metabolize adenosine into adenine 
. This may contribute to the inability of the mutant hearts to maintain normal contractile function; an idea which is in accordance with previous research suggesting that the failing heart is energy-starved 
. It should be noted that these alterations seems to be selectively related to decreased cholinergic function, as the changes observed in the microarray experiments were not observed in isoproterenol-treated mice.
Significant alterations in substrate metabolism have been observed during the progression of heart failure and it is suggested that these changes contribute to cardiac remodeling and dysfunction observed during disease progression 
. Previous studies have shown that, in end stage heart failure, there is an increase in glucose oxidation coupled with a decrease in fatty acid oxidation and these changes in substrate utilization lead to adverse effects during late stage heart failure 
To further examine transcriptional alterations in VAChT mutant hearts that may not have been identified in the microarray, we chose genes related to the lipid biosynthetic pathway (ACLY, ACC and FAS). These pathways have been previously found to be altered in heart failure 
. In agreement with the notion in cardiac dysfunction these pathways may be altered, chronic treatment with the β-agonist isoproterenol, which mimics the sympathetic overactivation observed in several cardiac diseases, increased mRNA levels for ACLY, ACC and FAS several fold. We also found an increase in mRNA expression of ACLY, ACC and FAS in VAChT mutant mice, suggesting at least some similarities between autonomic imbalance due to decreased cholinergic tone and sympathetic overactivation. Although gene expression changes were confirmed for several genes involved in the generation of long-chain fatty acids in VAChT-mutant mice, the protein levels for these enzymes appeared to be unaltered, although we cannot discard the possibility that their turnover might be increased.
It is important to note that VAChT KDHOM
animals exhibit a global decrease in VAChT levels and, therefore, decreased cholinergic tone. This is significant because it has recently been proposed that cardiomyocytes possess the machinery (VAChT, ChAT and CHT1) for de novo
production of ACh 
and are able to synthesize and release this neurotransmitter. This non-neuronal ACh may then act in an autocrine/paracrine fashion to amplify neuronal cholinergic signaling 
. We have recently demonstrated that this non-neuronal cardiomyocyte release of ACh plays an important role in the protection of myocytes against isoproterenol-induced hypertrophy and that VAChT mutant mice are deficient in non-neuronal ACh secretion as well 
. We cannot discard the possibility that the gene alterations we uncovered here may, at least in part, be due to deficient ACh release from cardiomyocytes. Future studies will be necessary to specifically analyze the importance of this non-neuronal cholinergic system in myocytes and its contribution to the cardiac dysfunction observed after reduced cholinergic tone. This may provide an unanticipated mechanism by which non-neuronal ACh can play an important role in cardiac function.