Multiple pathological events in exercised dystrophic mdx mice are targeted by pentoxifylline: outcome of a large array of in vivo and ex vivo tests

Abstract

The phosphodiesterases inhibitor pentoxifylline gained attention for Duchenne muscular dystrophy therapy for its claimed anti-inflammatory, antioxidant, and antifibrotic action. A recent finding also showed that pentoxifylline counteracts the abnormal overactivity of a voltage-independent calcium channel in myofibers of dystrophic mdx mice. The possible link between workload, altered calcium homeostasis, and oxidative stress pushed toward a more detailed investigation. Thus a 4- to 8-wk treatment with pentoxifylline (50 mg x kg(-1) x day(-1) ip) was performed in mdx mice, undergoing or not a chronic exercise on treadmill. In vivo, the treatment partially increased forelimb strength and enhanced resistance to treadmill running in exercised animals. Ex vivo, pentoxifylline restored the mechanical threshold, an electrophysiological index of calcium homeostasis, and reduced resting cytosolic calcium in extensor digitorum longus muscle fibers. Mn quenching and patch-clamp technique confirmed that this effect was paralleled by a drug-induced reduction of membrane permeability to calcium. The treatment also significantly enhanced isometric tetanic tension in mdx diaphragm. The plasma levels of creatine kinase and reactive oxygen species were both significantly reduced in treated-exercised animals. Dihydroethidium staining, used as an indicator of reactive oxygen species production, showed that pentoxifylline significantly reduced the exercise-induced increase in fluorescence in the mdx tibialis anterior muscle. A significant decrease in connective tissue area and profibrotic cytokine transforming growth factor-beta(1) was solely found in tibialis anterior muscle. In both diaphragm and gastrocnemius muscle, a significant increase in neural cell adhesion molecule-positive area was instead observed. This data supports the interest toward pentoxifylline and allows insight in the level of cross talk between pathogenetic events in workloaded dystrophic muscle.

Effects of prednisolone on the dystrophin-associated proteins in the blood–brain barrier and skeletal muscle of dystrophic mdx mice

“…Extensor digitorum longus (EDL) muscles were removed and placed in muscle chambers for electrophysiological or contraction measurements. 41 Blood was obtained from the heart and plasma prepared for spectrophotometric determination of creatine kinase (CK) activity using a commercial kit (Sentinel, Farmalab, Italy). 36 This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.…”

“…36 The mechanical threshold was determined using a two microelectrode 'point' voltage clamp method. 36,41 In brief, the two microelectrodes (spaced about 50 mm) were inserted into the central region of a superficial fiber and continuously viewed using a stereomicroscope ( Â 100 magnification). Depolarizing command pulses of duration ranging from 500 to 5 ms (0.3 Hz) were given from the holding potential (H) of 90 mV until visible contraction.…”

“…The threshold membrane potential (V, in mV) was read on a digital sample-and-hold millivoltmeter for each fiber at the various pulse durations t (in ms); mean values at each t allowed the construction of a 'strengthduration' curve; fit of data points to a classical nonlinear least square algorithm allowed to calculate the rheobase voltage (R, in mV). 41 For contractile recordings, one tendon side of EDL was fixed to a force transducer (1N 300C-LR, Aurora Scientific Inc., Canada) and connected to interface and acquisition units (Dynamic Muscle Control Acquisition System vs 4.1.6, ASI), while the opposite site is fixed to a chamber hook. Electrical stimulation field was obtained via two axial platinum wires connected to a stimulator (LE 12406, 2Biological Instruments, VA, Italy).…”

Redox homeostasis and age‐related deficits in neuromuscular integrity and function

“…In addition, despite the causal role of aberrant redox homeostasis in the development of muscular dystrophy,112 early clinical trials using antioxidants such as vitamins B and E and penicillamine did not show any statistically significant clinical benefits 298. Similarly, use of pentoxifylline, a phosphodiesterase inhibitor with potent antioxidant and anti‐inflammatory activity, failed to provide any improvements on muscle strength and function in Duchenne muscular dystrophy patients,299 despite showing significant muscle strength restoration on mdx mice 300. The use of antioxidant therapies in muscular dystrophy has been described in a recent review 293…”

Pentoxifylline as a rescue treatment for DMD

“…6,7 Pentoxifylline (PTX) is a phosphodiesterase inhibitor with potential ability to counteract the complex pathology in DMD; it improves calcium homeostasis and diminishes inflammation, fibrosis, and oxidative stress. 8,9 Preclinical studies showed that PTX reduced muscle strength deterioration by 51% in the exercised mdx mouse. 10 The anti-inflammatory effect of PTX is mediated primarily through the inhibition of tumor necrosis factor-␣ (TNF-␣) production by adenylyl cyclase activation and increased cellular cyclic AMP.…”