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Background: Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3β) survival pathway. Methods and Results: Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 ± 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3β, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3β pathway changed significantly with mechanical unloading. Conclusion: The PKB/Akt/GSK-3β pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support.

Background: Investigations involving biopsies of human cardiac tissue often assume that myocardial samples from a specific location are representative of the entire heart. Hypothesis: There are significant regional differences in gene expression in the heart. Methods: We used two models. In the first model, seven whole human hearts were cut in 1-cm slices from apex to base and 11 distinct regions were sampled. Full thickness left ventricular tissue was further subdivided equally into an inner, outer, and middle region. In the second model, hearts were removed from adult Sprague-Dawley rats and were divided into 4 regions. Using species-specific quantitative reverse transcriptase-polymerase chain reaction, we measured transcript levels of myosin heavy chain β (MHC-β), glucose transporter 1 (GLUT 1), and atrial natriuretic factor (ANF) in tissue samples from both models. Results: In human heart, there were significant differences in transcript levels between regions. The following patterns could be recognized among the seven hearts. ANF expression was highest in the subendocardial region. MHC-β and GLUT 1 transcript levels were higher in the right ventricle than the left ventricle. As expected, ANF transcript levels were highest in the atria, where MHC-β and GLUT 1 expression was low. Analogous to the human studies, MHC-β and GLUT 1 transcript levels were low in rat atria as compared to ventricles. In rat heart, MHC-β expression was higher in the left ventricle than the right ventricle while GLUT 1 expression was not significantly different between ventricles. Conclusion: Despite the large variability in transcript levels among different regions in human hearts, certain patterns in gene expression emerged suggesting that different anatomical regions of the heart also differ in respect to gene expression.

Background: We have previously shown that several metabolic genes are downregulated in the failing human heart. We now tested the hypothesis that mechanical unloading might reverse this process. Methods: Clinical data and myocardial tissue were collected from 14 failing hearts paired for the time of implantation and explantation of a left ventricular assist device (LVAD) and compared to 10 non-failing hearts. Transcript levels for key regulators of energy metabolism and for atrial natriuretic factor (ANF) were measured by real-time quantitative RT-PCR. Results: The expression of the glucose transporter 1 and 4 (GLUT1, GLUT4), muscle carnitine palmitoyl transferase-1 (mCPT-1), and uncoupling protein 3 (UCP3) were downregulated by up to 80% in the failing heart. Although LVAD treatment improved clinical markers of heart failure (decrease of left ventricular diastolic dimension and normalization of serum sodium), only UCP3 expression reversed to non-failing transcript levels following mechanical unloading. Conclusions: LVAD treatment only partially reverses depressed metabolic gene expression in the failing human heart. Reversal of depressed UCP3 expression may be an important mechanism for reducing the formation of oxygen-derived free radicals. Further studies are necessary to define the effects of mechanical unloading on post-transcriptional mechanisms.

Anecdotal and clinical evidence suggests that mechanical unloading may restore cardiac function by inducing changes in the biological properties of the failing heart. This challenges the notion that the progression of end-stage heart failure is an irreversible process ending in either death of the patient or transplantation. Although it is still not clear how mechanical unloading of the failing heart improves cardiac function, the process likely involves adaptive responses of cardiac myocytes on the cellular, extracellular, and subcellular levels.

Introduction: There is still no evidence whether human peritoneal mesothelial cells (HPMC) from patients with end-stage renal failure are altered in cell viability or show a different pattern of the release of proinflammatory cytokines. Also the serum of patients with uremia may contain substances stimulating the cytokine release of HPMC. Study Design: The IL-1β-induced IL-6/IL-8 release of HPMC from healthy donors and from patients with end-stage renal disease (ESRD) were measured before the start of chronic peritoneal dialysis (PD) and during PD therapy. Additionally the influence of uremic and non-uremic serum on IL-6 and IL-8 release of normal HPMC was studied. Cell viability was assessed by MTT assay and by the measurement of intracellular ATP (chemoluminescence assay). HPMC were obtained from the following patient groups: (1) non-uremic control patients (n = 7); (2) patients with ESRD undergoing PD catheter implantation for the first time (n = 7), and (3) patients on PD undergoing catheter exchange for noninfectious reasons (n = 6). Pooled human serum from PD patients and normal controls were used for stimulation experiments. HPMC from different donors were grown to confluence (second passage) and then stimulated with IL-1β (1,000 pg/ml in M199) for 24 h. IL-6 and IL-8 concentrations were measured in the supernatant by ELISA. Additionally uremic and non-uremic sera were incubated with HPMC from normal donors for 24 h with a subsequent 24-hour IL-1β stimulation. Mesothelial cell protein mass was determined by the Bradford reagent. Results: Non-uremic patients and ESRD patients did not differ with regard to the global cell viability of HPMC according to MTT assay activity or the intracellular ATP concentration. However, HPMC from uremic patients produced more IL-8 on IL-1β stimulation than the non-uremic controls (group 2, 53.5 ± 15.7 pg/µg; group 3, 70.5 ± 27.3 pg/µg vs. group 1, 24.0 ± 11.8 pg/µg). HPMC from patients on chronic PD additionally released significantly more IL-6 (30.5 ± 13.8 pg/µg) on IL-1β stimulation than uremic patients before the onset of PD (6.2 ± 2.6 pg/µg; p < 0.01). Incubation of normal HPMC with the serum from uremic donors produced an enhanced stimulated IL-8 release compared to the exposition with normal control serum (50.6 ± 6.1 vs. 20.8 ± 2.9 pg/µg; p < 0.01). Conclusion: HPMC from uremic patients more readily release IL-8 on stimulation with IL-1β. On chronic PD treatment IL-6 release was further enhanced. Not further classified serum components in uremia also enhance IL-6 and IL-8 release of HPMC.

The use of bicarbonate dialysate and high-flux and reprocessed dialyzers has raised concerns about the reverse transfer of dialysate contaminants into the blood compartment. This in vitro study was performed to investigate the reverse transfer of soluble Pseudomonas aeruginosa bacterial products across a polyether sulfone (PES), a newly developed synthetic polymer dialyzer. In vitro dialysis was carried out at 37°C in a closed countercurrent recirculating loop dialysis circuit with a new PES dialyzer. An equal mixture of heparinized whole blood (from healthy volunteers) with pyrogen-free tissue culture medium was circulated in the blood compartment, and bicarbonate dialysate was circulated in the dialysate compartment. After 15 min of dialysis, the dialysate was challenged sequentially with 10–4, 10–3, and 10–2 dilutions of a P. aeruginosa culture supernatant. 1-ml samples were drawn from the blood compartment 5 and 15 min after each challenge and incubated upright at 37°C. At the end of 24 h, Triton X-100 was added, in order to extract total interleukin (IL) 6 and IL-8 production by the whole-blood mixture. These cytokines were measured by electrochemiluminescence assays. At dilutions of 10–4 and 10–3, the reverse transfer of soluble bacterial products across the dialyzer was negligible. Five and 15 min after contaminating the dialysate with the highest concentration (10–2 dilution), the increase in IL-6 production was 239 ± 170% (p = 0.06) and 886 ± 444% (p = 0.02), respectively. However, comparing the IL-6-inducing potency of the 10–2 bacterial supernatant dilution to the spontaneous IL-6 production in the blood compartment during dialysis with the same dilution of dialysate contaminant, there was a dramatic reduction in IL-6 production by 94 and 89% at 5 and 15 min, respectively. Similarly, 5 and 15 min after contaminating the dialysate with the 10–2 dilution, the increase in IL-8 production was 357 ± 147% (p = 0.07) and 630 ± 229% (p = 0.04), respectively. However, comparing the IL-8-inducing potency of the 10–2 bacterial supernatant dilution to the spontaneous IL-8 production in the blood compartment during dialysis with the same dilution of dialysate contaminant, there was a dramatic reduction in IL-8 production by 93 and 92% at 5 and 15 min, respectively. These results demonstrate that PES dialyzers markedly attenuate passage of cytokine-inducing substances from contaminated dialysate, using a method that detects the entire cytokine synthetic output in the blood compartment.