Angiotensin II regulation of skeletal muscle regeneration, growth and satellite cell function

Abstract

<p> Local renin-angiotensin systems (RASs) have been described in many mammalian tissues. However, the role of angiotensin II (Ang II) in skeletal muscle is poorly understood with initial reports suggesting it may function to regulate overload-induced hypertrophy. Therefore, the purpose of this thesis was to 1) investigate the potential that adult skeletal muscle and muscle stem cells possess a local RAS. 2) Describe its role in regulating skeletal muscle regeneration and growth following injury and 3) demonstrate its capacity to regulate muscle stem cell activity and myogenesis. We report that cultured primary and C2C12 myoblasts and myotubes possess a local Ang II signalling system evidenced by the differential expression of angiotensinogen, angiotensin converting enzyme (ACE), and both angiotensin type 1 and 2 (AT1, AT2) receptors. Interestingly, myoblasts demonstrated the capacity to produce Ang II in spite of lacking renin expression. Furthermore, angiotensin receptors demonstrated differential localization with AT1 associated with actin filaments in proliferating myoblasts, and localized to the nucleus in differentiated myotubes. We also report that a local angiotensin system is present in vivo and responsive to myotrauma as cardiotoxin injection (to induce muscle injury) resulted in the increased staining intensity of angiotensinogen and AT1 during myogenesis with a progressive downregulation throughout the regenerative timecourse. </p> <p> To investigate the effects of Ang II signalling blockade on muscle growth and regeneration we induced muscle injury in mice supplemented with captopril (ACE inhibitor) or mice devoid of the AT1 a receptor. Histological analysis revealed that ACE inhibition resulted in a decreased muscle fibre growth, increased proportion of small myofibres, an inability to accrete myonuclei and a robust hyperplasia of muscle fibres. Similarly, AT1 a receptor ablation resulted in decreased muscle fibre growth following injury suggesting that these effects are receptor specific. </p> <p> To investigate the mechanisms underlying these effects we assessed the role of Ang II in regulating muscle satellite cell function. In vitro experiments revealed that Ang II had the ability to regulate the early response of satellite cells to muscle injury by acting as a potent transcriptional activator of quiescent myoblasts and directing their subsequent migration. Furthermore, these migratory effects were mediated through an Ang 11-induced increase in matrix metalloproteinase 2 (MMP2) content and reorganization of the actin cytoskeleton. Interestingly, Ang II may also participate in the fusion of myoblasts as captopril treatment suppressed the expression of markers of differentiation (myogenin) and maintained the expression of markers of proliferation (Pax7, Myf5). In agreement with this, IHC analysis revealed that ACE inhibition also induced a strong trend for a decrease in the proportion of myogenin positive cells following injury. Collectively, these results implicate the activation of local Ang II signalling system as a pleiotropic regulator of skeletal muscle growth. </p>