Timeline of cardiac dystrophy in 3-18-month-old MDX mice.

The dystrophin-deficient (mdx) mouse remains the most commonly used model for Duchenne muscular dystrophy (DMD). Mdx mice show a predominantly covert cardiomyopathy, the hallmark of which is fibrosis. We compared mdx and normal mice at six ages (3, 6, 9, 12, 15, and 18 months) using in vivo assessment of cardiac function, selective collagen staining, and measures of TGF-ß mRNA, Evans blue dye infiltration, macrophage infiltration, and aortic wall thickness. Clear temporal progression was demonstrated, including early fragility of cardiomyocyte membranes, which has an unrelated impact on cardiac function but is associated with macrophage infiltration and fibrosis. Aortic wall thickness is less in older mdx mice. Mdx mice display impaired responses to inotropic challenge from a young age; this is indicative of altered adrenoreceptor function. We draw attention to the paradox of ongoing fibrosis in mdx hearts without a strong molecular signature (in the form of TGF-ß mRNA expression).

A novel and simple method for genotyping the mdx mouse using high-resolution melt polymerase chain reaction.

The mdx mouse mutation arises from a C-to-T point mutation, which terminates the translation of dystrophin and results in the loss of a functional dystrophin protein. mdx mice are used widely in studies of the role of dystrophin and of potential treatments for Duchenne muscular dystrophy, thus accurate genotyping is essential. Current methods require labor-intensive efforts and can often lead to misconstrued results. This study describes a simple and highly reliable, sensitive, and user-friendly, high-resolution melt (HRM) assay that is able to utilize DNA obtained from a variety of sources in order to genotype the known sequence variant of the mdx mouse. Muscle Nerve 39: 603-608, 2009.

Long-term administration of antisense oligonucleotides into the paraspinal muscles of mdx mice reduces kyphosis.

The mdx mouse model of muscular dystrophy has a premature stop codon preventing production of dystrophin. This results in a progressive phenotype causing centronucleation of skeletal muscle fibers, muscle weakness, and fibrosis and kyphosis. Antisense oligonucleotides alter RNA splicing to exclude the nonsense mutation, while still maintaining the open reading frame to produce a shorter, but partially functional dystrophin protein that should ameliorate the extent of pathology. The present study investigated the benefits of chronic treatment of mdx mice by once-monthly deep intramuscular injections of antisense oligonucleotides into paraspinal muscles. After 8 mo of treatment, mdx mice had reduced development of kyphosis relative to untreated mdx mice, a benefit that was retained until completion of the study at 18 mo of age (16 mo of treatment). This was accompanied by reduced centronucleation in the latissimus dorsi and intercostals muscles and reduced fibrosis in the diaphragm and latissimus dorsi. These benefits were accompanied by a significant increase in dystrophin production. In conclusion, chronic antisense oligonucleotide treatment provides clear and ongoing benefits to paralumbar skeletal muscle, with associated marked reduction in kyphosis.

Long-term administration of pirfenidone improves cardiac function in mdx mice.

Duchenne muscular dystrophy, an X-linked recessive neuromuscular disorder due to lack of the protein dystrophin, manifests as progressive muscle degeneration and cardiomyopathy with increased fibrosis. The exact mechanisms involved in fibrosis are unknown, but a cytokine, transforming growth factor-beta (TGF-beta), is a likely mediator. This study tested whether the TGF-beta antagonist, pirfenidone, could reduce cardiac fibrosis. Eight-month-old mdx mice were treated for 7 months with 0.4%, 0.8%, and 1.2% pirfenidone in drinking water; untreated water was given to control mdx and C57 mice. Mice treated with 0.8% and 1.2% pirfendone had lowered cardiac TGF-beta mRNA and improved in vitro cardiac contractility (P < 0.05) to levels consistent with C57 mice, yet without a change in cardiac stiffness or fibrosis. These results show that the TGF-beta antagonist, pirfenidone, can improve cardiac function in mdx mice, potentially providing a new avenue for developing cardiac therapies for patients with Duchenne muscular dystrophy.

Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle.

The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium (P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria (P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates (P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels (P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation (P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.

Influence of age and moderate-intensity exercise training on heart rate variability in young and mature adults.

The purpose of this study was to examine the influence of age and moderate-intensity exercise training on heart rate variability (HRV), and to elucidate further the mechanism of training-induced bradycardia and cardioprotection. Electrocardiograms were recorded from 12 young (18-24 yrs) and 12 mature (29-43 yrs) individuals during supine rest and submaximal moderate exercise. Recordings were obtained prior to, midway, and following 16 weeks of aerobic exercise training designed to improve cardiorespiratory fitness and health. Training resulted in augmented estimated VO2max and bradycardia during rest and submaximal exercise. Total and low frequency components of HRV during exercise were significantly increased for the mature subjects following training whereas other measures of HRV were not significantly changed for either group. It was concluded that training of moderate intensity was insufficient to induce changes in the autonomic control of heart rate for young to mature subjects. The lack of significant HRV changes may suggest the existence of a vagal critical point, below which training-induced increases in vagal modulation may be forthcoming, and above which changes in vagal modulation may be negligible. Training-induced bradycardia and the cardioprotective effect of regular aerobic exercise may result from factors other than an increased vagal modulation.

Influence of intensive cycling training on heart rate variability during rest and exercise.

The current study examined whether changes in heart rate variability (HRV) following intensive cycling training contribute to the mechanism of training-induced bradycardia. Thirteen healthy untrained subjects, ages 18-27 years, underwent recordings of heart rate (HR) and VO2max before and after 8 weeks of cycling, 25-60 min/day, 5 days/week at > 80% maximum HR (HRmax). Heart rate recordings were obtained during supine rest and submaximal exercise and were analysed for the following components of HRV: low frequency (LF, 0.041-0.15 Hz); high frequency (HF, 0.15-0.40 Hz); LF/HF ratio and total power (TP, 0-0.40 Hz). At posttraining, VO2max was significantly increased while HR was significantly reduced at rest and all absolute exercise work rates. Training-induced lower HR was accompanied by significantly greater HF and TP during rest as well as LF, HF, and TP during all absolute exercise work rates. Posttraining HR and the majority of HRV measures were similar to pretraining values at the same relative exercise intensity (% HRmax). These results indicated that 8 weeks of intensive cycling training increased HRV and cardiac vagal modulation during rest and absolute exercise work rates but had little effect during relative exercise work rates. Increased vagal modulation resulting from intensive exercise training may contribute to the mechanism of training-induced bradycardia.

The streptozotocin-diabetic rat as a model of the chronic complications of human diabetes.

BACKGROUND: Diabetes in humans induces chronic complications such as cardiovascular damage, cataracts and retinopathy, nephropathy and polyneuropathy. The most common animal model of human diabetes is streptozotocin (STZ)-induced diabetes in the rat. METHODS: This project assessed cardiovascular, ocular and neuropathic changes over a period of 24 weeks post STZ administration in rats. RESULTS: STZ-diabetic rats (n = 96) showed stable signs of diabetes (hyperglycaemia, increased water and food intake with no increase in bodyweight): 52% of untreated STZ-diabetic rats (n = 50) survived 24 weeks after STZ administration. STZ-diabetic rats were normotensive with slowly developing systolic and diastolic dysfunction and an increased ventricular stiffness. Ventricular action potential durations were markedly prolonged. STZ-diabetic rats developed stable tactile allodynia. Cataracts developed to presumed blindness at 16 weeks but proliferative retinopathy was not observed even after 24 weeks. CONCLUSION: The chronic STZ-diabetic rat mimics many but not all of the chronic complications observed in the diabetic human. The chronic STZ-diabetic rat may be a useful model to test therapeutic approaches for amelioration of chronic diabetic complications in humans.