The Implications of Altered Cholinergic Signaling in Cardiac Health and Disease

­Cardiac remodeling and dysfunction occur prior to the onset of heart failure. Altered regulation of cardiac function by the autonomic nervous system has been implicated in the progression of heart disease. Both altered sympathetic and parasympathetic tone contribute to cardiac disease; however, the role of the parasympathetic nervous system, and specifically acetylcholine (ACh), in cardiac dysfunction has not been fully elucidated. In these studies, we sought to determine whether changes in neuronal and/or non-neuronal ACh release regulate cardiac activity and alter the progression of cardiac remodeling and dysfunction. A systemic decrease in the expression of the vesicular acetylcholine transporter (VAChT), the protein responsible for packaging ACh, led to the development of significant ventricular dysfunction coupled with significant transcriptional changes in cardiac tissue. Furthermore, we identified that murine cardiomyocytes possess an intrinsic cholinergic system, which prevents hypertrophy and molecular remodeling in cardiomyocytes in response to hyperadrenergic stimulation, in vitro. In addition, this cardiac non-neuronal cholinergic system (NNCS) is also critical in regulating heart activity and remodeling, in vivo. Inhibition of cardiomyocyte-specific ACh secretion led to delayed heart rate recovery following physiological stress, including exercise, as well as significant ventricular remodeling. Cardiomyocytes lacking the intrinsic cholinergic system displayed hypertrophy and molecular remodeling. This NNCS also plays a significant role under pathological conditions as chronic treatment with angiotensin II led to enhanced cardiac remodeling and ventricular dysfunction in mice lacking the NNCS. Additionally, this intrinsic cholinergic system in the heart is also present in human cardiomyocytes, suggesting the conserved expression of prototypic markers of the cholinergic system in man. This system might be of functional significance in cardiac disease as failing human cardiomyocytes exhibit increased VAChT expression, which likely leads to an increase in ACh secretion directly from cardiomyocytes. The increase in VAChT expression may play a protective role in heart failure, as overexpression of VAChT in mice did not reveal adverse phenotypes under physiological conditions. Our data suggest that both neuronal and non-neuronal ACh are critical in maintaining cardiac homeostasis and deficient cholinergic signaling contributes to ventricular remodeling and cardiac dysfunction

Cardiac acetylcholine inhibits ventricular remodeling and dysfunction under pathologic conditions

Autonomic dysfunction is a characteristic of cardiac disease and decreased vagal activity is observed in heart failure. Rodent cardiomyocytes produce de novo ACh, which is critical in maintaining cardiac homeostasis. We report that this nonneuronal cholinergic system is also found in human cardiomyocytes, which expressed choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). Furthermore, VAChT expression was increased 3- and 1.5-fold at the mRNA and protein level, respectively, in ventricular tissue from patients with heart failure, suggesting increased ACh secretion in disease. Weusedmicewith geneticdeletionof cardiomyocytespecificVAChTor ChATandmice overexpressingVAChT to test the functional significance of cholinergic signaling. Mice deficient for VAChT displayed an 8% decrease in fractional shortening and 13% decrease in ejection fraction comparedwith angiotensin II (Ang II)-treated control animals, suggesting enhanced ventricular dysfunction and pathologic remodeling in response to Ang II. Similar results were observed in ChAT-deficient mice. Conversely, no decline in ventricular function was observed in Ang II-treated VAChT overexpressors. Furthermore, the fibrotic area was significantly greater (P \u3c 0.05) in Ang II- treated VAChT-deficient mice (3.61 ± 0.64%) compared with wild-type animals (2.24±0.11%). In contrast, VAChT overexpressing mice didnot display an increase in collagen deposition. Our results provide new insight into cholinergic regulation of cardiac function, suggesting that a compensatory increase in cardiomyocyte VAChT levels may help offset cardiac remodeling in heart failure

Forebrain Cholinergic Signaling Regulates Innate Immune Responses and Inflammation

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNF alpha) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer\u27s disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation

Forebrain Cholinergic Signaling Regulates Innate Immune Responses and Inflammation

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation

An Analysis of the Myocardial Transcriptome in a Mouse Model of Cardiac Dysfunction with Decreased Cholinergic Neurotransmission

Autonomic dysfunction is observed in many cardiovascular diseases and contributes to cardiac remodeling and heart disease. We previously reported that a decrease in the expression levels of the vesicular acetylcholine transporter (VAChT) in genetically-modified homozygous mice (VAChT KDHOM) leads to decreased cholinergic tone, autonomic imbalance and a phenotype resembling cardiac dysfunction. In order to further understand the molecular changes resulting from chronic long-term decrease in parasympathetic tone, we undertook a transcriptome-based, microarray-driven approach to analyze gene expression changes in ventricular tissue from VAChT KDHOM mice. We demonstrate that a decrease in cholinergic tone is associated with alterations in gene expression in mutant hearts, which might contribute to increased ROS levels observed in these cardiomyocytes. In contrast, in another model of cardiac remodeling and autonomic imbalance, induced through chronic isoproterenol treatment to increase sympathetic drive, these genes did not appear to be altered in a pattern similar to that observed in VAChT KDHOM hearts. These data suggest the importance of maintaining a fine balance between the two branches of the autonomic nervous system and the significance of absolute levels of cholinergic tone in proper cardiac function

Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines

Hypertrophic cardiomyopathy is a common cause of mortality in congenital heart disease (CHD). Many gene abnormalities are associated with cardiac hypertrophy, but their function in cardiac development is not well understood. Loss-of-function mutations in PTPN11, which encodes the protein tyrosine phosphatase (PTP) SHP2, are implicated in CHD and cause Noonan syndrome with multiple lentigines (NSML), a condition that often presents with cardiac hypertrophic defects. Here, we found that NSML-associated hypertrophy stems from aberrant signaling mechanisms originating in developing endocardium. Trabeculation and valvular hyperplasia were diminished in hearts of embryonic mice expressing a human NSML-associated variant of SHP2, and these defects were recapitulated in mice expressing NSML-associated SHP2 specifically in endothelial, but not myocardial or neural crest, cells. In contrast, mice with myocardial- but not endothelial-specific NSML SHP2 expression developed ventricular septal defects, suggesting that NSML-associated mutations have both cell-autonomous and nonautonomous functions in cardiac development. However, only endothelial-specific expression of NSML-associated SHP2 induced adult-onset cardiac hypertrophy. Further, embryos expressing the NSML-associated SHP2 mutation exhibited aberrant AKT activity and decreased downstream forkhead box P1 (FOXP1)/FGF and NOTCH1/EPHB2 signaling, indicating that SHP2 is required for regulating reciprocal crosstalk between developing endocardium and myocardium. Together, our data provide functional and disease-based evidence that aberrant SHP2 signaling during cardiac development leads to CHD and adult-onset heart hypertrophy

There are no alterations in the protein levels of enzymes involved in lipid biosynthesis.

<p>Immunoblotting analysis of ATP citrate lyase (ACLY, <b>panel a</b>), Acetyl-CoA carboxylase (ACC; <b>panel b</b>) and fatty acid synthase (FAS; <b>panel c</b>) revealed no differences in the protein levels of these enzymes in VAChT KD^HOM mice. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.</p

VAChT KD^HOM cardiomyocytes show increased levels of ROS.

<p>Isolated cardiomyocytes loaded with a MitoSOX superoxide indicator reveal greater ROS levels in mutant myocytes (sample image; <b>panel a</b>). A robust, significant increase in fluorescence was observed in the KD cardiomyocytes as compared to wild-type control cells (<b>panel b</b>). Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice. Scale bar = 10 µm.</p

Purine nucleoside phosphorylases are upregulated in the hearts of VAChT KD^HOM mice.

<p>mRNA expression of purine nucleoside phosphorylase (Pnp; <b>panel a</b>) and purine nucleoside phosphorylase 2 (Pnp2, <b>panel b</b>) were upregulated. Pnp/Pnp2 protein content appears to upregulated in VAChT KD^HOM animals as compared to wild-type mice (<b>panel c</b>). Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.</p