Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure.

AIMS: Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. METHODS AND RESULTS: Study 1: homoarginine deficiency-female AGAT-/- and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT-/- had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6-7 weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency-as before, but AGAT-/- mice were given creatine-supplemented diet until 1 week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. CONCLUSIONS: Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Influence of homoarginine on creatine accumulation and biosynthesis in the mouse.

Organisms obtain creatine from their diet or by de novo synthesis via AGAT (L-arginine:glycine amidinotransferase) and GAMT (Guanidinoacetate N-methyltrasferase) in kidney and liver, respectively. AGAT also synthesizes homoarginine (hArg), low levels of which predict poor outcomes in human cardiovascular disease, while supplementation maintains contractility in murine heart failure. However, the expression pattern of AGAT has not been systematically studied in mouse tissues and nothing is known about potential feedback interactions between creatine and hArg. Herein, we show that C57BL/6J mice express AGAT and GAMT in kidney and liver respectively, whereas pancreas was the only organ to express appreciable levels of both enzymes, but no detectable transmembrane creatine transporter (Slc6A8). In contrast, kidney, left ventricle (LV), skeletal muscle and brown adipose tissue must rely on creatine transporter for uptake, since biosynthetic enzymes are not expressed. The effects of creatine and hArg supplementation were then tested in wild-type and AGAT knockout mice. Homoarginine did not alter creatine accumulation in plasma, LV or kidney, whereas in pancreas from AGAT KO, the addition of hArg resulted in higher levels of tissue creatine than creatine-supplementation alone (P < 0.05). AGAT protein expression in kidney was downregulated by creatine supplementation (P < 0.05), consistent with previous reports of end-product repression. For the first time, we show that hArg supplementation causes a similar down-regulation of AGAT protein (P < 0.05). These effects on AGAT were absent in the pancreas, suggesting organ specific mechanisms of regulation. These findings highlight the potential for interactions between creatine and hArg that may have implications for the use of dietary supplements and other therapeutic interventions.

BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes.

RATIONALE: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. OBJECTIVE: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. METHODS AND RESULTS: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31phosphorous magnetic resonance spectroscopy), and myocardial function. CONCLUSIONS: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.