Proteomic Correlates of the Urinary Protein/Creatinine Ratio in Heart Failure With Preserved Ejection Fraction.

Proteinuria is common in heart failure with preserved ejection fraction (HFpEF), but its biologic correlates are poorly understood. We assessed the relation between 49 plasma proteins and the urinary protein/creatinine ratio (UPCR) in 365 participants in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial. Linear regression and network analysis were used to represent relations between protein biomarkers and UPCR. Higher UPCR was associated with older age, a greater proportion of female gender, smaller prevalence of previous myocardial infarction, and greater prevalence of diabetes, insulin use, smoking, and statin use, in addition to a lower estimated glomerular filtration rate, hematocrit, and diastolic blood pressure. Growth differentiation factor 15 (GDF-15; ß = 0.15, p <0.0001), followed by N-terminal proatrial natriuretic peptide (NT-proANP; ß = 0.774, p <0.0001), adiponectin (ß = 0.0005, p <0.0001), fibroblast growth factor 23 (FGF-23, ß = 0.177; p <0.0001), and soluble tumor necrosis factor receptors I (ß = 0.002, p <0.0001) and II (ß = 0.093, p <0.0001) revealed the strongest associations with UPCR. Network analysis showed that UPCR is linked to various proteins primarily through FGF-23, which, along with GDF-15, indicated node characteristics with strong connectivity, whereas UPCR did not. In a model that included FGF-23 and UPCR, the former was predictive of the risk of death or heart-failure hospital admission (standardized hazard ratio 1.83, 95% confidence interval 1.49 to 2.26, p <0.0001) and/or all-cause death (standardized hazard ratio 1.59, 95% confidence interval 1.22 to 2.07, p = 0.0005), whereas UPCR was not prognostic. Proteinuria in HFpEF exhibits distinct proteomic correlates, primarily through its association with FGF-23, a well-known prognostic marker in HFpEF. However, in contrast to FGF-23, UPCR does not hold independent prognostic value.

Impact of diabetes on remodelling, microvascular function and exercise capacity in aortic stenosis.

OBJECTIVE: To characterise cardiac remodelling, exercise capacity and fibroinflammatory biomarkers in patients with aortic stenosis (AS) with and without diabetes, and assess the impact of diabetes on outcomes. METHODS: Patients with moderate or severe AS with and without diabetes underwent echocardiography, stress cardiovascular magnetic resonance (CMR), cardiopulmonary exercise testing and plasma biomarker analysis. Primary endpoint for survival analysis was a composite of cardiovascular mortality, myocardial infarction, hospitalisation with heart failure, syncope or arrhythmia. Secondary endpoint was all-cause death. RESULTS: Diabetes (n=56) and non-diabetes groups (n=198) were well matched for age, sex, ethnicity, blood pressure and severity of AS. The diabetes group had higher body mass index, lower estimated glomerular filtration rate and higher rates of hypertension, hyperlipidaemia and symptoms of AS. Biventricular volumes and systolic function were similar, but the diabetes group had higher extracellular volume fraction (25.9%±3.1% vs 24.8%±2.4%, p=0.020), lower myocardial perfusion reserve (2.02±0.75 vs 2.34±0.68, p=0.046) and lower percentage predicted peak oxygen consumption (68%±21% vs 77%±17%, p=0.002) compared with the non-diabetes group. Higher levels of renin (log10renin: 3.27±0.59 vs 2.82±0.69 pg/mL, p<0.001) were found in diabetes. Multivariable Cox regression analysis showed diabetes was not associated with cardiovascular outcomes, but was independently associated with all-cause mortality (HR 2.04, 95% CI 1.05 to 4.00; p=0.037). CONCLUSIONS: In patients with moderate-to-severe AS, diabetes is associated with reduced exercise capacity, increased diffuse myocardial fibrosis and microvascular dysfunction, but not cardiovascular events despite a small increase in mortality.

A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension.

Genetic variants in the third intron of the PRDM6 gene have been associated with BP traits in multiple GWAS. By combining fine mapping, massively parallel reporter assays, and gene editing, we identified super enhancers that drive the expression of PRDM6 and are partly regulated by STAT1 as the causal variants for hypertension. The heterozygous disruption of Prdm6 in mice expressing Cre recombinase under the control of mouse smooth muscle cell protein 22-α promoter (Prdm6fl/+ SM22-Cre) exhibited a markedly higher number of renin-producing cells in the kidneys at E18.5 compared with WT littermates and developed salt-induced systemic hypertension that was completely responsive to the renin inhibitor aliskiren. Strikingly, RNA-Seq analysis of the mouse aortas identified a network of PRDM6-regulated genes that are located in GWAS-associated loci for blood pressure, most notably Sox6, which modulates renin expression in the kidney. Accordingly, the smooth muscle cell-specific disruption of Sox6 in Prdm6fl/+ SM22-Cre mice resulted in a dramatic reduction of renin. Fate mapping and histological studies also showed increased numbers of neural crest-derived cells accompanied by increased collagen deposition in the kidneys of Prdm6fl/+ Wnt1Cre-ZsGreen1Cre mice compared with WT mice. These findings establish the role of PRDM6 as a regulator of renin-producing cell differentiation into smooth muscle cells and as an attractive target for the development of antihypertensive drugs.

TCF7L2 transcriptionally regulates Fgf15 to maintain bile acid and lipid homeostasis through gut-liver crosstalk.

FGF19/FGF15 is an endocrine regulator of hepatic bile salt and lipid metabolism, which has shown promising effects in the treatment of NASH in clinical trials. FGF19/15 is transcribed and released from enterocytes of the small intestine into enterohepatic circulation in response to bile-induced FXR activation. Previously, the TSS of FGF19 was identified to bind Wnt-regulated TCF7L2/encoded transcription factor TCF4 in colorectal cancer cells. Impaired Wnt signaling and specifical loss of function of its coreceptor LRP6 have been associated with NASH. We, therefore, examined if TCF7L2/TCF4 upregulates Fgf19 in the small intestine and restrains NASH through gut-liver crosstalk. We examined the mice globally overexpressing, haploinsufficient, and conditional knockout models of TCF7L2 in the intestinal epithelium. The TCF7L2+/- mice exhibited increased plasma bile salts and lipids and developed diet-induced fatty liver disease while mice globally overexpressing TCF7L2 were protected against these traits. Comprehensive in vivo analysis revealed that TCF7L2 transcriptionally upregulates FGF15 in the gut, leading to reduced bile synthesis and diminished intestinal lipid uptake. Accordingly, VilinCreert2 ; Tcf7L2fl/fl mice showed reduced Fgf19 in the ileum, and increased plasma bile. The global overexpression of TCF7L2 in mice with metabolic syndrome-linked LRP6R611C substitution rescued the fatty liver and fibrosis in the latter. Strikingly, the hepatic levels of TCF4 were reduced and CYP7a1 was increased in human NASH, indicating the relevance of TCF4-dependent regulation of bile synthesis to human disease. These studies identify the critical role of TCF4 as an upstream regulator of the FGF15-mediated gut-liver crosstalk that maintains bile and liver triglyceride homeostasis.

Wnt Signaling Cascades and Their Role in Coronary Artery Health and Disease.

The Wnt signaling is classified as two distinct pathways of canonical Wnt/ß-catenin signaling, and the non-canonical pathways of planar cell polarity and Wnt/Ca2+ pathways. However, the scientific discoveries in recent years have shown that canonical and non-canonical Wnts pathways are intertwined and have complex interaction with other major signaling pathways such as hedgehog, Hippo and TOR signaling. Wnt signaling plays important roles in cell proliferation, differentiation and migration during embryonic development. The impairment of these pathways during embryonic development often leads to major congenital defects. In adult organisms Wnt expression is more restricted to proliferating tissues, where it plays a key role in tissue regeneration. In addition, the disruption of homeostatic processes of multicellular organisms may give rise to reactivation and/or altered activation of Wnt signaling, leading to development of malignant tumors and chronic diseases such as type-2 diabetes and adult cardiovascular diseases. Coronary artery disease (CAD) is the leading cause of death in the world. The disease is the consequences of two distinct disease processes: Atherosclerosis, a primarily inflammatory disease and plaque erosion, a disease process associated with endothelial cell defect and smooth muscle proliferation with only modest contribution of inflammatory cells. The atherosclerosis is itself a multifactorial disease that is initiated by lipid deposition and endothelial dysfunction, triggering vascular inflammation via recruitment and aggregation of monocytes and their transformation to foam cell by the uptake of modified low-density lipoprotein (LDL), culminating in an atheromatous plaque core formation. Further accumulation of lipids, infiltration and proliferation of vascular smooth muscle cells (VSMCs) and extracellular matrix deposition result in intimal hyperplasia. Myocardial infarction is the ultimate consequence of these processes and is caused by plaque rupture and hypercoagulation. In vivo studies have established the role of the Wnt pathway in all phases of atherosclerosis development, though much remains unknown or controversial. Less is known about the mechanisms that induce plaque erosion. The limited evidence in mouse models of Wnt coreceptor LRP6 mutation and heterozygous TCF7L2 knock out mice implicate altered Wnt signaling also in the pathogenesis of plaque erosion. In this article we focus and review the role of the Wnt pathway in CAD pathophysiology from clinical and experimental standpoints.

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

The vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vri Δ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vri Δ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

VRILLE Controls PDF Neuropeptide Accumulation and Arborization Rhythms in Small Ventrolateral Neurons to Drive Rhythmic Behavior in Drosophila.

In Drosophila, the circadian clock is comprised of transcriptional feedback loops that control rhythmic gene expression responsible for daily rhythms in physiology, metabolism, and behavior. The core feedback loop, which employs CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors to drive rhythmic transcription peaking at dusk, is required for circadian timekeeping and overt behavioral rhythms. CLK-CYC also activates an interlocked feedback loop, which uses the PAR DOMAIN PROTEIN 1ε (PDP1ε) activator and the VRILLE (VRI) repressor to drive rhythmic transcription peaking at dawn. Although Pdp1ε mutants disrupt activity rhythms without eliminating clock function, whether vri is required for clock function and/or output is not known. Using a conditionally inactivatable transgene to rescue vri developmental lethality, we show that clock function persists after vri inactivation but that activity rhythms are abolished. The inactivation of vri disrupts multiple output pathways thought to be important for activity rhythms, including PDF accumulation and arborization rhythms in the small ventrolateral neuron (sLNv) dorsal projection. These results demonstrate that vri acts as a key regulator of clock output and suggest that the primary function of the interlocked feedback loop in Drosophila is to drive rhythmic transcription required for overt rhythms.

Drosophila CRY Entrains Clocks in Body Tissues to Light and Maintains Passive Membrane Properties in a Non-clock Body Tissue Independent of Light.

Circadian (∼24 hr) clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor CRYPTOCHROME (CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading TIMELESS (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, we generated a GFP-tagged-cry transgene that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.

Phosphorylation of the transcription activator CLOCK regulates progression through a ∼ 24-h feedback loop to influence the circadian period in Drosophila.

Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.