Andrew Hoey

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‐β), is a likely mediator. This study tested whether the TGF‐β 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‐β 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‐β antagonist, pirfenidone, can improve cardiac function in mdx mice, potentially providing a new avenue for developing cardiac therapies for patients with Duchenne muscular dystrophy. Muscle Nerve, 2006

Improved cardiovascular function with aminoguanidine in DOCA-salt hypertensive rats

1 The ability of aminoguanidine (AG), an inhibitor of collagen crosslinking, to prevent changes in cardiac and vascular structure and function has been determined in the deoxycorticosterone acetate (DOCA)‐salt hypertensive rat as a model of the cardiovascular remodelling observed in chronic human hypertension. 2 Uninephrectomized rats (UNX) administered DOCA (25u2003mg every fourth day s.c.) and 1% NaCl in drinking water for 28 days developed cardiovascular remodelling shown as systolic hypertension, left ventricular hypertrophy, increased thoracic aortic and left ventricular wall thickness, increased left ventricular inflammatory cell infiltration together with increased interstitial collagen and increased passive diastolic stiffness, impaired contractility, prolongation of the action potential duration and vascular dysfunction. 3 Treatment with AG (0.05–0.1% in drinking water; average 182±17u2003mgu2003kg−1u2003day−1 in DOCA‐salt rats) decreased blood pressure (DOCA‐salt 176±4; +AG 144±5u2003mmHg; *P<0.05 vs DOCA‐salt), decreased left ventricular wet weights (DOCA‐salt 3.17±0.07; +AG 2.66±0.08u2003mgu2003g−1 body wt*), reduced diastolic stiffness constant (DOCA‐salt 30.1±1.2; +AG 24.3±1.2* (dimensionless)), improved cardiac contractility (DOCA‐salt 1610±130; +AG 2370±100u2003mmHgu2003s−1*) and vascular reactivity (3.4‐fold increase in maximal contractile response to noradrenaline, 3.2‐fold increase in maximal relaxation response to acetylcholine, twofold increase in maximal relaxation response to sodium nitroprusside) and prolonged the action potential duration at 50% repolarization without altering collagen content or inflammatory cell infiltration. 4 Thus, cardiovascular function in DOCA‐salt hypertensive rats can be improved by AG independent of changes in collagen content. This suggests that collagen crosslinking is an important cause of cardiovascular dysfunction during cardiovascular remodelling in hypertension.

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‐β 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‐β mRNA expression). Muscle Nerve, 2010

Reversal of cardiac dysfunction by selective ET-A receptor antagonism

The effectiveness of a selective endothelin receptor‐A (ET‐A) antagonist, A‐127722 (approximately 10u2003mgu2003kg−1u2003day−1 as 200u2003mgu2003kg−1 powdered food), to reverse existing cardiac remodelling and prevent further remodelling was tested in deoxycorticosterone acetate (DOCA)‐salt hypertensive rats. Uninephrectomised rats (UNX) administered DOCA (25u2003mg every fourth day s.c.) and 1% NaCl in drinking water for 28 days developed hypertension (systolic blood pressure (BP): UNX 128±6u2003mmHg, DOCA‐salt 182±5*u2003mmHg; *P<0.05 vs UNX), left ventricular hypertrophy (UNX 1.99±0.06u2003mgu2003kg−1 body wt, DOCA‐salt 3.30±0.08*u2003mgu2003kg−1 body wt), decreased left ventricular internal diameter (UNX 6.69±0.18u2003mm, DOCA‐salt 5.51±0.37*u2003mm), an increased left ventricular monocyte/macrophage infiltration together with an increased interstitial collagen from 2.7±0.3 to 11.7±1.3%, increased passive diastolic stiffness (UNX 21.1±0.5, DOCA‐salt 30.1±1.3*), prolongation of the action potential duration at 20 and 90% of repolarisation (APD20–UNX 6.8±1.1, DOCA‐salt 10.1±1.5*u2003ms; APD90–UNX 34.4±3.5u2003ms, DOCA‐salt 64.3±10.4*u2003ms) and vascular dysfunctions (2.6‐fold decrease in maximal contractile response to noradrenaline, 3.5‐fold decrease in maximal relaxation response to acetylcholine). Administration of A‐127722 for 14 days starting 14 days after surgery attenuated the increases in systolic BP (150±6**u2003mmHg, **P<0.05 vs DOCA‐salt), left ventricular wet weight (2.65±0.06**u2003mgu2003kg−1 body wt) and internal diameter (6.39±0.31**u2003mm), prevented left ventricular monocyte/macrophage accumulation, attenuated the increased left ventricular interstitial collagen (7.6±1.3%**), reversed the increased passive diastolic stiffness (22.1±1.2**), attenuated the action potential duration prolongation (APD20 – 7.6±1.4**, APD90 – 41.5±6.9**u2003ms) and normalised changes in vascular function. ET‐A receptor antagonism both reverses and prevents the cardiac and vascular remodelling in DOCA‐salt hypertension and improves cardiovascular function.

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.

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

Rosuvastatin attenuates heart failure and cardiac remodelling in the ageing spontaneously hypertensive rat.

3-hydroxy-3-methylglutaryl(HMG)-Coenzyme(Co)A reductase inhibitors such as rosuvastatin may improve clinical status in patients with hypertension and heart failure. The ageing spontaneously hypertensive rat (SHR) closely mimics the chronic heart failure disease process observed in humans. This study examined the structural and functional changes in the cardiovascular system of 15-month-old SHR and normotensive Wistar-Kyoto (WKY) rats treated with rosuvastatin (20u2003mg/kg/day perorally) for 24u2003weeks. Cardiovascular structure and function were monitored serially by echocardiography. At 21u2003months, ex vivo Langendorff, electrophysiological or histological studies were performed. Chronic rosuvastatin treatment attenuated elevations of left ventricular wet weight (mg/g body weight: 21-month WKY, 2.30u2003±u20030.04; 15-month SHR, 3.03u2003±u20030.08; 21-month SHR, 4.09u2003±u20030.10; 21-month SHRu2003+u2003rosuvastatin, 3.50u2003±u20030.13), myocardial extracellular matrix content (% left ventricular area: 21-month WKY, 7.6u2003±u20030.5; 15-month SHR, 13.2u2003±u20030.8; 21-month SHR 19.6u2003±u20031.0; 21-month SHR with rosuvastatin 14.6u2003±u20031.2) and diastolic stiffness (κ: 21-month WKY, 24.9u2003±u20030.6; 15-month SHR, 26.4u2003±u20030.4; 21-month SHR, 33.1u2003±u20030.8; 21-month SHRu2003+u2003rosuvastatin, 27.5u2003±u20030.6) as well as attenuating the deterioration of systolic and diastolic function (fractional shortening %: 21-month WKY, 66u2003±u20032; 15-month SHR, 51u2003±u20033; 21-month SHR, 38u2003±u20033; 21-month SHRu2003+u2003rosuvastatin, 52u2003±u20034). There was no effect on the increased systolic blood pressure, plasma low-density lipoprotein concentrations or the prolonged action potential duration. Thus, chronic rosuvastatin treatment may attenuate myocardial dysfunction in heart failure by preventing fibrosis.

Attenuation of cardiovascular remodeling in DOCA-salt rats by the vasopeptidase inhibitor, omapatrilat.

Omapatrilat, a vasopeptidase inhibitor, inhibits both neutral endopeptidase and angiotensin-converting enzyme with similar potency. The aim of this study was to investigate whether omapatrilat prevents or reverses cardiovascular remodeling and hypertension in deoxycorticosterone acetate (DOCA)-salt rats. Male Wistar rats (313 ± 2 g, n=114) were uninephrectomized (UNX) with or without further treatment with DOCA and 1% NaCl in the drinking water. Compared with UNX control rats, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, perivascular and interstitial cardiac fibrosis and inflammation, endothelial dysfunction, and the prolongation of ventricular action potential duration within four weeks. The administration of omapatrilat (40 mg/kg/day po) for two weeks commencing two weeks after surgery attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation. In contrast, omapatrilat treatment did not lower systolic blood pressure nor improve endothelial dysfunction. This study concludes that the renin-angiotensin-aldosterone, natriuretic peptide, and bradykinin systems are directly involved in the pathogenesis of cardiovascular remodeling in the DOCA-salt model of hypertension in rats, which may be independent of their effects on blood pressure.

Alterations in cardiac dihydropyridine receptors and calcium channel function in mdx mice

The efficacy of antioxidant supplementation in the prevention of cardiovascular disease appears equivocal, however the use of more potent antioxidant combinations than those traditionally used may exert a more positive effect. We have shown previously that supplementation of vitamin E and α-lipoic acid increases cardiac performance during post-ischemia reperfusion in older rats and increases Bcl-2 levels in endothelial cells. The purpose of this study was to examine the effects of vitamin E and α-lipoic acid supplementation on myocardial gene expression with a view to determine their mechanism of action. Young male rats received either a control (n=7) or vitamin E and α-lipoic acid supplemented diet (n=8) for 14 weeks. RNA from myocardial tissue was then amplified and samples were pooled within groups and competitively hybridized to 8.5K oligonucleotide rat microarrays. The relative expression of each gene was then compared to the control sample. Animals that received the antioxidant-supplemented diet exhibited upregulation (>1.5×) of 13 genes in the myocardium with 2 genes downregulated. Upregulated genes include those involved in cell growth and maintenance (LynB, Csf1r, Akt2, Tp53), cell signaling (LynB, Csf1r) and signal transduction (Pacsin2, Csf1r). Downregulated genes encode thyroid (Thrsp) and F-actin binding proteins (Nexilin).Duchenne muscular dystrophy (DMD) is a fatal neuromuscular condition affecting approximately one in 3500 live male births resulting from the lack of the myocyte protein dystrophin. The absence of dystrophin in cardiac myocytes is associated with calcium overload which in turn activates calcium-dependent proteolytic enzymes contributing to congestive heart failure, muscle necrosis and fibrosis. To date, the basis for the calcium overload has not been determined. Since L-type calcium channels are a major mediator of calcium influx we determined their potential contribution to the calcium overload. Male muscular dystrophy (mdx) mice and control C57BL10ScSn (C57) mice aged 12– 16 weeks were used in all experiments. In tissue bath studies, isolated contracting left atria from mdx revealed a reduced potency to the dihydropyridine (DHP) agonist BayK8644 and antagonist nifedipine (P < 0.05). Similarly, radioligand binding studies using the DHP antagonist [3H]-PN 200-110 showed a reduced potency (P < 0.05) in isolated membranes, associated with an increased receptor density (P < 0.05). The increased receptor density was supported by RT-PCR experiments revealing increased RNAfor the DHP receptor. Patch clamp studies revealed the presence of a diltiazem sensitive calcium current that showed delayed inactivation in isolated mdx myocytes (P < 0.01). In conclusion, the increased number of DHP binding sites and the delay in L-type current inactivation may both contribute to increased calcium influx and hence calcium overload in the dystrophin deficient mdx cardiac myocytes.