Cellular mechanisms of acute renal failure in rats

Groeneveld, A.B.J. [Promotor]Tangelder, G.J. [Promotor]Sipkema, P. [Copromotor]Musters, R.J.P. [Copromotor

SIH--a novel lipophilic iron chelator--protects H9c2 cardiomyoblasts from oxidative stress-induced mitochondrial injury and cell death

Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, iron chelation may be an effective therapeutic approach, but the progress in this area is hindered by the lack of effective agents. In this study, using the rat heart myoblast-derived cell line H9c2, we aimed to investigate whether the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) protects the cells against hydrogen peroxide (H2O2)-induced cytotoxicity. Exposure of cells to 100 μmol/l H2O2 has within 4 h induced a complete dissipation of their mitochondrial membrane potential (ΔΨm) . Co-treatment with SIH dose-dependently reduced (EC50 = 0.8 μmol/l) or even completely abolished (3 μmol/l) this collapse. Furthermore, the latter SIH concentration was capable to fully prevent alterations in cell morphology, and inhibited both apoptosis (annexin-V staining, nuclear chromatin shrinkage, TUNEL positivity) and necrosis (propidium iodide staining), even 24 h after the H2O2 exposure. In comparison, deferoxamin (a commercially available hydrophilic iron chelator used in clinical practice and most previous studies) was cytoprotective only at three-order higher and clinically unachievable concentrations (EC50 = 1300 μmol/l). Thus, in this study, we present iron chelation as a very powerful tool by which oxidative stress-induced myocardial damage can be prevented

Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4-and 70-kDa dextrans through the cytosol, and localization of 155-and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin-and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4-to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size. (Circ Res. 2009; 104: 679-687.

Enhanced myofilament responsiveness upon β-adrenergic stimulation in post-infarct remodeled myocardium.

AbstractPreviously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed β-adrenergic receptor (β-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (Fmax) and higher Ca2+-sensitivity in MI compared to sham. Fmax did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca2+-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of β-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca2+-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise