Empagliflozin restores chronic kidney disease-induced impairment of endothelial regulation of cardiomyocyte relaxation and contraction

Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodium-hydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability

High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes

The overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (−13% and −19%, respectively) as was the decay of cytosolic calcium during diastole (−15% and −21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/β-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD

High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes

\u3cp\u3eThe overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (−13% and −19%, respectively) as was the decay of cytosolic calcium during diastole (−15% and −21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/β-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD.\u3c/p\u3

Contribution of Impaired Parasympathetic Activity to Right Ventricular Dysfunction and Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension

BACKGROUND: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). METHODS: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (alpha-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. RESULTS: Patients with PAH with lower RV ejection fraction (<41%) had a significantly reduced heart rate recovery in comparison with patients with higher RV ejection fraction. In PAH RV samples, alpha-7 nicotinic acetylcholine receptor was increased and acetylcholinesterase activity was reduced versus controls. No difference in muscarinic acetylcholine type 2 receptor expression was observed. Chronic PYR treatment in PH rats normalized the cardiovascular autonomic function, demonstrated by an increase in parasympathetic activity and baroreflex sensitivity. PYR improved survival, increased RV contractility, and reduced RV stiffness, RV hypertrophy, RV fibrosis, RV inflammation, and RV alpha-7 nicotinic acetylcholine receptor and muscarinic acetylcholine type 2 receptor expression, as well. Furthermore, PYR reduced pulmonary vascular resistance, RV afterload, and pulmonary vascular remodeling, which was associated with reduced local and systemic inflammation. CONCLUSIONS: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.Science Without Borders grant, Conselho Nacional de Desenvolvimento Cientifico e TecnologicoVICI grant from the Netherlands Organization for Scientific Research (NWO)Sao Paulo Research FoundationNetherlands CardioVascular Research Initiative grantVENI grant from NWOL'Oreal/UNESCO for Women in ScienceNetherlands Institute for Advanced Studies (NIAS)American Thoracic Society (ATS: Jerry Wojciechowski Memorial Pulmonary Hypertension Research Grant)European Respiratory SocietyInstitute for Cardiovascular Research (ICaR-VU)Vrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Pulmonol, Amsterdam, NetherlandsVrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Physiol, Amsterdam, NetherlandsVrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Cardiol, Amsterdam, NetherlandsLeiden Univ, Med Ctr, Dept Mol Cell Biol, Lab Expt Cardiol, Leiden, NetherlandsUniv Fed Sao Paulo, Inst Sci & Technol, Sao Paulo, BrazilUniv Sydney, Heart & Lung Transplant Unit, St Vincents Hosp, Sydney, NSW, AustraliaUniv Sydney, Bosch Inst, Sydney, NSW, AustraliaUniv Paris Saclay, Univ Paris Sud, Le Kremlin Bicetre, FranceINSERM, UMR S 999, Le Plessis Robinson, FranceUniv Fed Sao Paulo, Inst Sci & Technol, Sao Paulo, BrazilCNPq-Brasil-245849/2012-2FAPESP-2016/08263-9Netherlands CardioVascular Research Initiative grant: CVON 2012-08VENI grant from NWO: 916.14.099Web of Scienc

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring–based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARɣ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ~250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNnA binding model for PPARɣ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARɣ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARɣ copy number