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.

Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity.

Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.

Nitric oxide synthase regulation of cardiac excitation-contraction coupling in health and disease.

Significant advances in our understanding of the ability of nitric oxide synthases (NOS) to modulate cardiac function have provided key insights into the role NOS play in the regulation of excitation-contraction (EC) coupling in health and disease. Through both cGMP-dependent and cGMP-independent (e.g. S-nitrosylation) mechanisms, NOS have the ability to alter intracellular Ca(2+) handling and the myofilament response to Ca(2+), thereby impacting the systolic and diastolic performance of the myocardium. Findings from experiments using nitric oxide (NO) donors and NOS inhibition or gene deletion clearly implicate dysfunctional NOS as a critical contributor to many cardiovascular disease states. However, studies to date have only partially addressed NOS isoform-specific effects and, more importantly, how subcellular localization of NOS influences ion channels involved in myocardial EC coupling and excitability. In this review, we focus on the contribution of each NOS isoform to cardiac dysfunction and on the role of uncoupled NOS activity in common cardiac disease states, including heart failure, diabetic cardiomyopathy, ischemia/reperfusion injury and atrial fibrillation. We also review evidence that clearly indicates the importance of NO in cardioprotection. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

Daily running enhances molecular and physiological circadian rhythms in skeletal muscle.

OBJECTIVE: Exercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise. METHODS: Wildtype C57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise. RESULTS: We demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed in mice, and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the murine muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the time-dependent response to moderate daily exercise in mice. CONCLUSIONS: Collectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of murine muscle gene expression, which in turn support daily fluctuations in exercise performance.

Clocking In, Working Out: Circadian Regulation of Exercise Physiology.

Research over the past century indicates that the daily timing of physical activity impacts on both immediate performance and long-term training efficacy. Recently, several molecular connections between circadian clocks and exercise physiology have been identified. Circadian clocks are protein-based oscillators that enable anticipation of daily environmental cycles. Cell-autonomous clocks are present in almost all cells of the body, and their timing is set by a variety of internal and external signals, including hormones and dietary intake. Improved understanding of the relationship between molecular clocks and exercise will benefit professional athletes and public health guidelines for the general population. We discuss here the role of circadian clocks in exercise, and explore time-of-day effects and the proposed molecular and physiological mechanisms.

Phosphorylation of CRY1 Serine 71 Alters Voluntary Activity but Not Circadian Rhythms In Vivo.

Circadian clocks allow organisms to anticipate repetitive changes in their environment such as food availability, temperature, and predation. While they most clearly manifest at the behavioral level, driving sleep-wake cycles, for example, they also provide critical temporal regulation at the level of individual tissues. Circadian clocks within organs act to ensure that each tissue is functioning in a coordinated manner to anticipate the needs of the organism as a whole but also allow for adaptation of organs to their local environment. One critical aspect of this environment is energy availability, which is communicated at the cellular level via changes in metabolites such as ATP, calcium, and NADH. AMP-activated protein kinase (AMPK) is both sensitive to fluctuations in secondary metabolites and capable of resetting the circadian clock via destabilization of the core clock components CRY and PER. Phosphorylation of serine 71 of CRY1 by AMPK destabilizes CRY1 by decreasing its interaction with binding partner PER2, thus enabling greater association with the SCF complex substrate adaptor FBXL3. Here, we describe a transgenic mouse harboring germline mutation of CRY1 serine 71 to alanine. Unexpectedly, this mutation does not affect the steady-state level of CRY1 protein in mouse livers or quadriceps. We also did not detect changes in either behavioral or molecular circadian rhythms, but female Cry1S71A mice exhibit decreased voluntary locomotor activity compared with wild-type littermates. Together, these findings suggest that phosphorylation of CRY1 serine 71 is not required for the regulation of circadian rhythms under normal physiological conditions. However, it may be involved in responding to metabolic challenges or in other aspects of physiology that contribute to voluntary activity levels.

The Liver Circadian Clock Modulates Biochemical and Physiological Responses to Metformin.

Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Although metformin is a relatively safe and effective drug, its clinical efficacy is variable and under certain circumstances it may contribute to life-threatening lactic acidosis. Thus, additional understanding of metformin pharmacokinetics and pharmacodynamics could provide important information regarding therapeutic use of this widely prescribed drug. Here we report a significant effect of time of day on acute blood glucose reduction in response to metformin administration and on blood lactate levels in healthy mice. Furthermore, we demonstrate that while metformin transport into hepatocytes is unaltered by time of day, the kinetics of metformin-induced activation of AMP-activated protein kinase (AMPK) in the liver are remarkably altered with circadian time. Liver-specific ablation of Bmal1 expression alters metformin induction of AMPK and blood glucose response but does not completely abolish time of day differences. Together, these data demonstrate that circadian rhythms affect the biological responses to metformin in a complex manner.

CRY1/2 Selectively Repress PPARδ and Limit Exercise Capacity.

Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator-activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1-/-;Cry2-/- myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ, and thereby alter energy storage and substrate selection for energy production.

Tetrahydrobiopterin Protects Against Hypertrophic Heart Disease Independent of Myocardial Nitric Oxide Synthase Coupling.

BACKGROUND: Nitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin (BH4)-a cofactor required for normal nitric oxide synthase function-supporting a pathophysiological link. Genetically augmenting BH4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH4 administration. We tested whether the primary cellular target of BH4 is the cardiomyocyte or whether other novel mechanisms are invoked. METHODS AND RESULTS: Mice with cardiomyocyte-specific overexpression of GTP cyclohydrolase 1 (mGCH1) and wild-type littermates underwent transverse aortic constriction. The mGCH1 mice had markedly increased myocardial BH4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction-induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction. CONCLUSIONS: BH4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.