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The calcium/calmodulin-dependent phosphatase calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro. Treatment with the antineoplastic agent doxorubicin (DOX) was shown to activate calcineurin signaling in H9c2 rat cardiac muscle cells; however, the effect of this activation on hypertrophy was not investigated. Therefore, the present study was undertaken to examine the involvement of calcineurin activation in DOX-induced cardiac cell hypertrophy. H9c2 cells were treated with 1 M DOX for 2 h following pretreatment with and in the presence of calcineurin inhibitors cyclosporine A (CsA) or FK506 (tacrolimus). Subsequent analysis of calcineurin signaling and cellular hypertrophy was performed 8 to 48 h after the treatment. DOX treatment activated calcineurin signaling and resulted in cellular hypertrophy as assessed by an increase in cell volume and protein content per cell. Inhibition of calcineurin with CsA or FK506 blocked DOX-induced calcineurin signaling. However, this inhibition did not prevent the DOX-induced hypertrophic response in H9c2 cells. Further evaluation of the possible signaling pathways involved in DOX-induced H9c2 cellular hypertrophy revealed that DOX treatment resulted in phosphorylation of the serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K). Moreover, the DOXinduced hypertrophic response was blunted by LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a specific inhibitor for PI3K. These results demonstrate that, although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced hypertrophy in H9c2 cells. Rather, the PI3K-Akt signaling pathway seems to be more critically involved in DOX-induced hypertrophy.

IntroductionMethicillin-resistant S. aureus (MRSA) is a common cause of bone and joint infection. BT2-peg2-vancomycin is an investigational bone-targeted formulation of vancomycin which we hypothesized would have increased antimicrobial activity compared to conventional vancomycin in a chronic experimental MRSA osteomyelitis model.MethodsWe tested bone affinity using an hydroxyapatite (HA) binding assay, assessed the in vitro antimicrobial susceptibility of 30 MRSA isolates, and compared vancomycin and BT2-peg2-vancomycin in a rat experimental osteomyelitis model.ResultsVancomycin did not bind to hydroxyapatite (HA binding index = 0), whereas BT2-peg2-vancomycin showed appreciable binding (HA binding index = 57). The MIC50 was 1 μg/ml and the MIC90 was 2 μg/ml for both vancomycin and BT2-peg2-vancomycin. The MBC90 was 16 and 4 μg/ml for vancomycin and BT2-peg2-vancomycin, respectively. Treatment with 50 mg/kg of vancomycin every 12 hours (median, 4.73 log10 cfu/g), 63.85 mg/kg (equivalent to 50 mg/kg vancomycin) of BT2-peg2-vancomycin every 12 hours (median, 3.93 log10 cfu/g) or 63.85 mg/kg of BT2-peg2-vancomycin once per week (median, 5.00 log10 cfu/g) was more active than no treatment (median, 5.22 log10 cfu/g) (P =0.0481). Treatment with 63.85 mg/kg of BT2-peg2-vancomycin every 12 hours was more active than all other treatment regimens evaluated (P≤0.0150), but was associated with high plasma BT2-peg2-vancomycin levels, decreased animal weight, increased kidney size, creatinine and BUN, and leukocytosis with tubulointerstitial nephritis.ConclusionWith optimization of pharmacokinetic parameters to prevent toxicity, BT2-peg2-vancomycin may be useful in the treatment of MRSA osteomyelitis.

Previous studies using a cardiac-specific metallothionein (MT)-overexpressing transgenic (MT-TG) mouse model have demonstrated that MT protects from doxorubicin (DOX)-induced oxidative heart injury. The molecular mechanisms that underlie this cardioprotection, however, have yet to be defined. In the present study, we tested the hypothesis that MT overexpression activates cytoprotective mechanisms, leading to cardiac protection from DOX toxicity. MT-TG mice and nontransgenic wild-type (WT) controls were treated i.p. with DOX at a single dose of 20 mg/kg and sacrificed on the third day after the treatment. An expression proteomic analysis involving twodimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify MT-induced changes in cytoprotection-related proteins. We identified 18 proteins that were modified by DOX treatment in the heart. These proteins included those involved in cellular antioxidant defense, enzymes of the mitochondrial electron transport chain, enzymes involved in ␤-oxidation of fatty acids and glycolysis, and proteins involved in regulation of cardiac muscle contraction. However, the most dominant modification by MT is the cytochrome c oxidase subunit Va (CCOVa). In response to DOX treatment, a specific isoform of CCO-Va was enhanced in the MT-TG but not in the WT mouse hearts. Because CCO-Va is a critical component in the mitochondrial electron transport chain, the results suggest that the cardioprotective effect of MT may be related to an increased expression or a differential modification of CCO-Va.

hWe examined the pharmacokinetic properties of vancomycin conjugated to a bone-targeting agent (BT) with high affinity for hydroxyapatite after systemic intravenous administration. The results confirm enhanced persistence of BT-vancomycin in plasma and enhanced accumulation in bone relative to vancomycin. This suggests that BT-vancomycin may be a potential carrier for the systemic targeted delivery of vancomycin in the treatment of bone infections, potentially reducing the reliance on surgical debridement to achieve the desired therapeutic outcome. Osteomyelitis is defined as any inflammatory process in bone, the most common cause of which is infection. Although many bacterial pathogens have been associated with osteomyelitis, Staphylococcus aureus is the predominant cause and the pathogen responsible for the most serious forms of bone infection (1). Given the increasing prevalence of S. aureus strains resistant to methicillin (2), vancomycin remains the most commonly used antibiotic for the treatment of these infections (3). While true vancomycin resistance is rare, S. aureus strains with reduced susceptibility are common and often arise as a consequence of the prolonged periods of vancomycin therapy required to treat bone infections (1, 4). Vancomycin acts by inhibiting bacterial cell wall biosynthesis (5, 6) and is a large hydrophilic molecule that has limited penetration into bone and therefore low bone bioavailability when administered systemically (7). These factors emphasize the need to develop methods to enhance delivery of vancomycin to bone in the treatment of osteomyelitis. One way to accomplish this is to employ local antibiotic delivery, which while useful suffers from inherent limitations, not the least being the ability to gain direct access to the infection site (8-16). Thus, one of the major challenges to improve therapeutic outcomes for osteomyelitis patients is to develop methods for the systemic deliv-