Masseter Muscle Myofibrillar Protein Synthesis and Degradation in an Experimental Critical Illness Myopathy Model

Abstract

Critical illness myopathy (CIM) is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A) ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU) model, where animals were exposed to sedation, neuromuscular blockade (NMB), mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs). Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model.

The Sick and the Weak: Neuropathies/Myopathies in the Critically Ill

“…In contrast to limb muscles, the masseter showed only a mild and delayed decline in myosin-to-actin ratio, smaller reduction in fiber size, and no transcriptional downregulation of myofibrillar protein synthesis. In contrast to both limb muscles and the diaphragm, there was an increase in MuRF1 levels and more sustainable levels of sarcomere protective HSPs and autophagy (8). Thus the better preserved myofibrillar protein expression and muscle fiber size in the masseter muscle compared with limb muscles in response to the ICU condition were the result of muscle-specific differences in both transcriptional regulation of myofibrillar protein synthesis and protein degradation pathways and coupled to a decreased activation of the IGF-I/PI3K/Akt pathway as well as protective effects of HSP and autophagy machineries (8).…”

“…The exact mechanism underlying the relative sparing of craniofacial muscles versus limb muscles, which is also reproduced in the porcine animal model, is not known, but intrinsic differences related to differences in embryonic origin and inherent differences in membrane properties and myofibrillar protein expression may have a significant effect on muscle-specific differences also in mechanosensation and responsiveness to mechanical silencing (1,8).…”

“…Thus this model offers a unique possibility to conduct time-resolved analyses with a high temporal resolution on the effects of the ICU condition on muscle, muscle specific differences, as well as in the design and evaluation of specific intervention strategies (395). In accordance with clinical and experimental studies using the porcine ICU model, mechanical ventilation and immobilization are required for durations longer than 5 days to induce the CIM phenotype, i.e., muscle wasting and a preferential myosin loss in limb muscles, relative sparing of carniofacial muscles, and severe negative effects on regulation of diaphragm muscle fiber function (8,133,517,520). The CIM phenotype was observed in the rat model in the absence of both systemic corticosteriod hormone administration and sepsis.…”

“…Despite these disadvantages, the in vivo experimental animal ICU models presented here (porcine and rat) are best at modeling triggers for CIM in patients. These models also do an excellent job at modeling pathological changes such as (FIGURE 14) 1) severe atrophy (8, 133, 510 -512, 517), 2) preferential and significant myosin loss (510 -512, 517, 574), 3) sarcomere disorganization (FIGURE 14), 4) electrical hypoexcitability (511,517), and 5) inadequate autophagy activation (8,31). …”

Masticatory muscles of mouse do not undergo atrophy in space

“…Further, in clinical situations where there is severe muscle wasting, as seen in patients with acute quadriplegic myopathy in the intensive care unit, the masticatory muscles are spared. This suggests these muscles are equipped with a different load sensing program than limb muscles (20,21). Animal models for acute quadriplegic myopathy recapitulate the protection against atrophy in MA muscles in stark contrast to the muscle atrophy in the rest of the body (22,23).…”