Atrial fibrillation burden and subsequent heart failure events in patients with cardiac resynchronization therapy devices.

BACKGROUND: Atrial fibrillation (AF) and heart failure (HF) often coexist but little is known on how AF burden associates with subsequent episodes of HF. OBJECTIVE: The aim of this study was to quantitatively assess the short- and long-term association of AF burden with subsequent episodes of HF events in patients with reduced ejection fraction. METHODS: Patients with cardiac resynchronization therapy (CRT) devices with at least 90 days of device data were included in the study. Time-dependent Cox regression with a 7-day window was used to evaluate the association of short- and long-term AF burden with subsequent HF events. Each patient with HF was matched to two control patients without an HF event based on age, gender, year of implant and CRT defibrillation capability. RESULTS: In our cohort with 2:1 matching (N = 549), 183 patients developed HF events and 275 (50.1%) had AF over an average follow-up of 24 ± 11 months. A 1-hour increase in short-term AF burden was associated with a 3% increased risk of HF events (HR, 1.034; 95% confidence interval [CI], 1.012-1.056; P = .01; HR for 24-hour = 2.23). In contrast, the association between long-term AF burden and subsequent HF events was not statistically significant (HR, 1.009; 95% CI, 0.992-1.026; P = .373). CONCLUSION: A 24-hour increase in AF burden is associated with a more than two-fold increased risk of HF events over the subsequent week while the long-term AF burden is not significantly associated with HF events.

Real-world behavior of CRT pacing using the AdaptivCRT algorithm on patient outcomes: Effect on mortality and atrial fibrillation incidence.

BACKGROUND: The AdaptivCRT (aCRT) algorithm continuously adjusts cardiac resynchronization therapy (CRT) according to intrinsic atrioventricular conduction, providing synchronized left ventricular pacing in patients with normal PR interval and adaptive BiV pacing in patients with prolonged PR interval. Previous analyses demonstrated an association between aCRT and clinical benefit. We evaluated the incidence of patient mortality and atrial fibrillation (AF) with aCRT compared with standard CRT in a real-world population. METHODS AND RESULTS: Patients enrolled in the Medtronic Personalized CRT Registry and implanted with a CRT from 2013-2018 were divided into aCRT ON or standard CRT groups based upon device-stored data. A Frailty survival model was used to evaluate the potential survival benefit of aCRT, accounting for patient heterogeneity and center variability. Daily AF burden and first device-detected AF episodes of various durations were recorded by the device during follow-up. A total of 1814 CRT patients with no reported long-standing AF history at implant were included. Mean follow-up time was 26.1 ± 16.5 months and 1162 patients (64.1%) had aCRT ON. Patient survival probability at 36 months was 88.3% for aCRT ON and 83.7% for standard CRT (covariate-adjusted hazard ratio [HR] = 0.71, 95% CI: 0.53-0.96, P = .028). Mean AF burden during follow-up was consistently lower in aCRT ON patients compared with standard CRT. At 36 months, the probability of AF was lower in patients with aCRT ON, regardless of which AF definition threshold was applied (6 minutes-30 days, all P < .001). CONCLUSION: Use of the AdaptivCRT algorithm was associated with improved patient survival and lower incidence of AF in a real-world, prospective, nonrandomized registry.

Cardiac hypertrophy is enhanced in PPAR alpha-/- mice in response to chronic pressure overload.

AIMS: Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPARalpha modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPARalpha aggravates the cardiac hypertrophic response to pressure overload. METHODS AND RESULTS: Male PPARalpha-/- and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPARalpha-/- than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPARalpha-/- mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, alpha-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-alpha, cyclo-oxygenase-2) marker genes were higher in PPARalpha-/- than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPARalpha-/- mice, but were not further compromised by TAC. CONCLUSION: The present findings show that the absence of PPARalpha results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPARalpha exerts salutary effects during cardiac hypertrophy.

Transcriptomic analysis of PPARalpha-dependent alterations during cardiac hypertrophy.

Peroxisome proliferator-activated receptor (PPAR)alpha regulates lipid metabolism at the transcriptional level and modulates the expression of genes involved in inflammation, cell proliferation, and differentiation. Although PPARalpha has been shown to mitigate cardiac hypertrophy, knowledge about underlying mechanisms and the nature of signaling pathways involved is fragmentary and incomplete. The aim of this study was to identify the processes and signaling pathways regulated by PPARalpha in hearts challenged by a chronic pressure overload by means of whole genome transcriptomic analysis. PPARalpha-/- and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days, and left ventricular gene expression profile was determined with Affymetrix GeneChip Mouse Genome 430 2.0 arrays containing >45,000 probe sets. In unchallenged hearts, the mere lack of PPARalpha resulted in 821 differentially expressed genes, many of which are related to lipid metabolism and immune response. TAC resulted in a more pronounced cardiac hypertrophy and more extensive changes in gene expression (1,910 and 312 differentially expressed genes, respectively) in PPARalpha-/- mice than in wild-type mice. Many of the hypertrophy-related genes were related to development, signal transduction, actin filament organization, and collagen synthesis. Compared with wild-type hypertrophied hearts, PPARalpha-/- hypertrophied hearts revealed enrichment of gene clusters related to extracellular matrix remodeling, immune response, oxidative stress, and inflammatory signaling pathways. The present study therefore demonstrates that, in addition to lipid metabolism, PPARalpha is an important modulator of immune and inflammatory response in cardiac muscle.

Activation of PPARdelta inhibits cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts.

OBJECTIVE: The development of heart failure is invariably associated with extensive fibrosis. Treatment with Peroxisome Proliferator-Activated Receptor (PPAR) ligands has been shown to attenuate cardiac fibrosis, but the molecular mechanism underlying this protective effect has remained largely unknown. In this study the potential of each PPAR isoform (PPARalpha, delta, and gamma) to attenuate cardiac fibroblast proliferation, fibroblast (CF) to myofibroblast (CMF) transdifferentiation, and collagen synthesis was investigated. METHODS AND RESULTS: PPARdelta was found to be the most abundant isoform in both CF and CMF. Only the PPARdelta ligand GW501516, but not PPARalpha ligand Wy-14,643 or PPARgamma ligand rosiglitazone, significantly increased PPAR-dependent promoter activity and expression of the PPAR-responsive gene UCP2 ( approximately 5-fold). GW501516 reduced the proliferation rate of CF (-38%) and CMF (-26%), which was associated with increased expression of the cell cycle inhibitor gene G0/G1 switch gene 2 (G0S2). Exposure of CF to the PPARdelta ligand or adenoviral overexpression of PPARdelta significantly decreased alpha-smooth muscle actin (alpha-SMA) levels, indicating a reduced CF to CMF transition. The inhibition of transdifferentiation by PPARdelta correlated with an increase in PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome ten) expression. (3)H-Proline incorporation assays demonstrated a GW501516 induced decline in collagen synthesis (-36%) in CF. CONCLUSION: Cardiac fibroblast proliferation, fibroblast to myofibroblast differentiation and collagen synthesis were reduced after activation of PPARdelta, suggesting that PPARdelta represents an attractive molecular target for attenuating cardiac fibrosis.

Metabolic remodelling of the failing heart: the cardiac burn-out syndrome?

It has been postulated that the failing heart suffers from chronic energy starvation, and that derangements in cardiac energy conversion are accessory to the progressive nature of this disease. The molecular mechanisms driving this 'metabolic remodelling' process and their significance for the development of cardiac failure are still open to discussion. Next to changes in mitochondrial function, the hypertrophied heart is characterized by a marked shift in substrate preference away from fatty acids towards glucose. It has been argued that the decline in fatty acid oxidation is not fully compensated for by a rise in glucose oxidation, thereby imposing an additional burden on overall ATP generating capacity. Several lines of evidence suggest that these metabolic adaptations are brought about, at least in part, by alterations in the rate of transcription of genes encoding for proteins involved in substrate transport and metabolism. Here, the principal metabolic changes are reviewed and the various molecular mechanisms that are likely to play a role are discussed. In addition, the potential significance of these changes for the aetiology of heart failure is evaluated.

Inflammatory pathways are activated during cardiomyocyte hypertrophy and attenuated by peroxisome proliferator-activated receptors PPARalpha and PPARdelta.

Accumulating evidence indicates an important role for inflammation in cardiac hypertrophy and failure. Peroxisome proliferator-activated receptors (PPARs) have been reported to attenuate inflammatory signaling pathways and, as such, may interfere with cardiac remodeling. Accordingly, the objectives of the present study were to explore the relationship between cardiomyocyte hypertrophy and inflammation and to investigate whether PPARalpha and PPARdelta are able to inhibit NF-kappaB activation and, consequently, the hypertrophic growth response of neonatal rat cardiomyocytes (NCM). mRNA levels of markers of both hypertrophy and inflammation were increased following treatment with the pro-hypertrophic factor phenylephrine (PE) or the chemokine TNF-alpha. Induction of inflammatory genes was found to be fast (within 2 h after stimulation) and transient, while induction of hypertrophic marker genes was more gradual (peaking at 24-48 h). Inflammatory and hypertrophic pathways appeared to converge on NF-kappaB as both PE and TNF-alpha increased NF-kappaB binding activity as measured by electrophoretic mobility shift assay. Following transient transfection, the p65-induced transcriptional activation of a NF-kappaB reporter construct was significantly blunted after co-transfection of PPARalpha or PPARdelta in the presence of their respective ligands. Finally, adenoviral overexpression of PPARalpha and PPARdelta markedly attenuated cell enlargement and the expression of hypertrophic marker genes in PE-stimulated NCM. The collective findings reveal a close relationship between hypertrophic and inflammatory signaling pathways in the cardiomyocyte. It was shown that both PPARalpha and PPARdelta are able to mitigate cardiomyocyte hypertrophy in vitro by inhibiting NF-kappaB activation.

Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites.

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism (K(m) of 1.5 +/- 0.3 mM, V(max) of 47 +/- 7 pmol x mg(-1) x min(-1), and Hill coefficient of 1.7 +/- 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 +/- 8 microM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity (K(m) and V(max) value of 180 +/- 20 microM and 58 +/- 4 pmol x mg(-1) x min(-1), respectively, Hill coefficient of 1.4 +/- 0.1) to single binding site kinetics (K(m) and V(max) value of 2.2 +/- 0.9 mM and 280 +/- 50 pmol x mg(-1) x min(-1), respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is also present in the luminal membrane of proximal tubule cells of the kidney, but little information is available on its role in the renal excretion of xenobiotics. The authors compared renal transport of the fluorescent Mrp2 substrates calcein, fluo-3, and lucifer yellow (LY) between perfused kidneys isolated from Wistar Hannover (WH) and TR(-) rats. Isolated rat kidneys were perfused with 100 nM of the nonfluorescent calcein-AM or 500 nM fluo3-AM, which enter the tubular cells by diffusion and are hydrolyzed intracellularly into the fluorescent anion. The urinary excretion rates of calcein and fluo-3 were 3 to 4 times lower in perfused kidneys from TR(-) rats compared with WH rats. In contrast, the renal excretion of LY (10 micro M, free anion) was somewhat delayed but appeared unimpaired in TR(-) rats. Membrane vesicles from Sf9 cells expressing human MRP2 or human MRP4 indicated that MRP2 exhibits a preferential affinity for calcein and fluo-3, whereas LY is a better substrate for MRP4. We conclude that the renal clearance of the Mrp2 substrates calcein and fluo-3 is significantly reduced in TR(-) rat; for LY, the absence of the transporter may be compensated for by (an)other organic anion transporter(s).

Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter.

p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.

Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule.

We recently demonstrated in isolated killifish renal proximal tubules that two classes of nephrotoxicants, aminoglycoside antibiotics and radiocontrast agents, rapidly decrease transport mediated by multidrug resistance protein 2 (Mrp2) by causing endothelin (ET) release and signaling through an ET(B) receptor and protein kinase C (PKC). In the present study, we used killifish proximal tubules, fluorescein methotrexate, a fluorescent model substrate for Mrp2, and confocal microscopy to examine the effects of two heavy metal salts (CdCl(2) and HgCl(2)) on Mrp2 function. Three patterns of effects were seen. First, exposing tubules to 10 microM CdCl(2) or 100 nM HgCl(2) for 30 min reduced Mrp2-mediated transport. This reduction was abolished by the ET(B) receptor antagonist, RES-701-1, and by the PKC-selective inhibitor, bis-indolylmaleimide I; neither of these pharmacological tools by itself affected transport. As with aminoglycoside antibiotics and radiocontrast agents, the acute effects of 10 microM CdCl(2) or 100 nM HgCl(2) on transport were also blocked by nifedipine, suggesting that Ca(2+) also initiated cadmium and mercury action. Second, exposure to higher concentrations of CdCl(2) and HgCl(2) appeared to be toxic. Third, exposing tubules for 6 to 24 h to lower levels of CdCl(2) increased Mrp2-mediated transport and Mrp2 immunostaining at the luminal membrane of the proximal tubule cells. Together, these findings indicate that exposure of renal proximal tubules to heavy metals initially leads to reduced Mrp2 function but is followed by an induction in Mrp2-mediated transport after long-term exposure.

The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP.

The cyclic nucleotides cAMP and cGMP play key roles in cellular signaling and the extracellular regulation of fluid balance. In the kidney, cAMP is excreted across the apical proximal tubular membrane into urine, where it reduces phosphate reabsorption through a dipyridamole-sensitive mechanism that is not fully understood. It has long been known that this cAMP efflux pathway is dependent on ATP and is inhibited by probenecid. However, its identity and whether cGMP shares the same transporter have not been established. Here the expression, localization, and functional properties of human multidrug resistance protein 4 (MRP4) are reported. MRP4 is localized to the proximal tubule apical membrane of human kidney, and membrane vesicles from Sf9 cells expressing human MRP4 exhibit ATP-dependent transport of [(3)H]cAMP and [(3)H]cGMP. Both probenecid and dipyridamole are potent MRP4 inhibitors. ATP-dependent [(3)H]methotrexate and [(3)H]estradiol-17beta-D-glucuronide transport by MRP4 and interactions with the anionic conjugates S-(2,4-dinitrophenyl)-glutathione, N-acetyl-(2,4-dinitrophenyl)-cysteine, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules.