|dc.description.abstract||Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that physically interact with and inhibit the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. Results from our laboratory and others have shown that, under conditions of high-fat feeding, SLN is recruited in skeletal muscles to uncouple SERCA-mediated Ca2+ transport from ATP hydrolysis thereby increasing energy expenditure by the SERCA pumps and combating obesity. Since, PLN is considered structurally and functionally homologous with SLN, we initially questioned whether PLN could also uncouple the SERCA pump, increase energy expenditure, and combat the obesigenic effects of a high-fat diet. To this end, we purchased commercially available mice that overexpressed PLN (PlnOE); however, the surprising discovery of pathology within their soleus muscles shifted the focus from metabolism to myopathy.
In the first study of this thesis, the overall objectives were to characterize the PlnOE mouse model with respect to SERCA and muscle function, and to diagnose the disease found in their skeletal muscles. Since the PlnOE mice were designed to overexpress PLN specifically in their type I skeletal muscle fibres, the analyses of this study was restricted to the postural muscles, soleus and gluteus minimus, that normally contain approximately 55-65% and 27% type I fibre proportions, respectively. As a result of the 6-to-7-fold upregulation in the inhibitory PLN protein, reductions in SERCA’s apparent affinity for Ca2+ (-0.16 KCa in pCa units, P < 0.05), maximal activity (-15-28%, P < 0.05), and Ca2+ uptake (-20-74%, P < 0.05) in the soleus and gluteus minimus muscles from PlnOE mice were observed. Reductions in the rates of relaxation (-dF/dt, -60%, P < 0.05) in isolated soleus muscles assessed in vitro further support the decrements in SERCA function. With respect to the myopathy, the soleus muscles exhibited muscle weakness and progressive atrophy, which was associated with an overall enhancement of all the major proteolytic pathways existing in skeletal muscle (caspase-3, cathepsin, calpain, and proteasome, P < 0.05) and oxidative stress revealed with dichlorofluoroscein fluorescence assays (P < 0.05) and protein nitrosylation (P < 0.05). Moreover, results from histological, histochemical, and immunofluorescent analyses of the PlnOE soleus and gluteus minimus muscles revealed greater central nuclei, type I fibre predominance and hypotrophy, and central aggregation of oxidative activity, suggesting that the myopathy found in these muscles resembled the rare congenital disease centronuclear myopathy (CNM). Interestingly, preliminary findings from muscle biopsies from three CNM patients and five healthy controls revealed that, in CNM, SERCA function is significantly impaired (-53% maximal activity, P < 0.05) and is associated with a trending increase in PLN expression (P = 0.08); however future experiments with more CNM patients that are genetically related are required to further examine PLN’s potential role in CNM. Nevertheless, the results from study I demonstrate, for the first time, the importance of PLN in skeletal muscle health and disease, and have uncovered a novel model for CNM to test novel mechanisms and therapeutic strategies.
In study II, the diaphragm muscles from PlnOE mice were examined because these types of investigations that assess respiratory function in animal models of CNM are limited. Since the diaphragm muscles typically comprise of 10% type I fibres, it was hypothesized that the diaphragm muscles would also display impaired SERCA function, muscle weakness, and CNM pathology. The results from this study; however, indicate that although SERCA’s affinity for Ca2+ was significantly lower in the PlnOE diaphragm compared with wild-type (-0.07 KCa in pCa units, P < 0.05), SERCA function was not impaired to the extent seen in the PlnOE soleus and gluteus minimus muscles with no differences in maximal SERCA activity and only a 15% reduction in Ca2+ uptake that did not reach statistical significance (P = 0.08). Moreover, there was very little pathology in the PlnOE diaphragm with no impairments in in vitro force contractility and only type I fibre hypotrophy being evident with no central nuclei, type I fibre predominance, or central aggregation of oxidative activity. Thus, the diaphragm muscles appear to confer resistance to PlnOE-induced myopathy and understanding the underlying mechanisms may lead to novel therapeutic strategies.
One interesting possibility that was tested in this thesis involved the potential role of SLN, which was upregulated according to disease severity in the PlnOE mice as SLN was increased 7-9 fold (P < 0.05) in the PlnOE gluteus minimus and soleus muscles; but only increased 2.5-fold (P < 0.05) in the PlnOE diaphragm. Similar patterns of SLN expression were previously observed in the dystrophic mdx and mdx/utrophin-null mouse models where SLN was found upregulated to a greater extent in the mdx/utrophin-null mouse which exhibits far worse dystrophic pathology. The question of whether this increase in SLN represents a maladaptive or adaptive role in myopathy was the focus of study III in this thesis. To this end, three separate mouse models of myopathy were tested.
First, the role of SLN in the PlnOE-induced myopathy was assessed by generating the PlnOE/SlnKO mouse. The results from these experiments show that in the absence of Sln the PlnOE induced myopathy was worsened with greater reductions in in vitro soleus force production and muscle mass relative to body weight (P < 0.05). The exacerbated muscle weakness and atrophy could not be attributed to differences in food intake, daily activity, or muscle proteolysis, and was instead accounted for by a failure to promote type II fibre hypertrophy. In response to Pln overexpression and the concomitant reduction in type I fibre cross-sectional area, the type II fibres hypertrophy (P < 0.05) and transition towards a slower oxidative fibre population (P < 0.05) – a result due to the increased load bearing activities of the type II fibres that subsequently activate the Ca2+-dependent serine/threonine phosphatase calcineurin. Without Sln, the PlnOE/SlnKO mice had lower activation of calcineurin revealed by greater levels of phosphorylated nuclear factor of activated T-cells (NFAT, P < 0.05) thereby causing a failure in the type II fibres to hypertrophy and transition to a slow fibre population. Interestingly, similar effects were observed in mechanical overload experiments where WT plantaris muscles exhibited ectopic SLN expression, myofibre hypertrophy and a transition towards a slow-oxidative population (P < 0.05) as a result of calcineurin activation (61% lower phosphorylation of NFAT, P < 0.05). In contrast, SlnKO plantaris muscles failed to hypertrophy and transition to a slow-oxidative phenotype as a result of a blunted activation of calcineurin (22% lower phosphorylation of NFAT, P = 0.11). Collectively, the results from these experiments suggest that the SLN upregulation in the PlnOE mouse is critical in triggering calcineurin and mediating adaptive muscle remodeling.
The second mouse model of disease used in study III was the tenotomized soleus, which is a model of hindlimb unloading known to cause muscle degeneration. The results from these experiments revealed that in the absence of a 14-fold upregulation of SLN, the soleus muscles exhibited greater muscle atrophy (P < 0.05), associated with larger percent reductions in myofibre cross-secitonal area (CSA) and an accelerated slow-to-fast fibre type transition. Consistent with SLN’s role in activating calcineurin, tenotomy resulted in a significantly lower phophorylation of NFAT in WT soleus (-57%, P < 0.03), which was blunted in the SlnKO soleus (-32%, P = 0.06). Taken together, these results suggests that SLN upregulation, as result of muscle unloading, is required to amplify calcineurin signaling to counteract/stabilize the effects of muscle atrophy and slow-to-fast fibre type transitions.
Finally, the role of SLN in the mdx mouse was determined by generating mdx/SlnKO mice. Consistent with the findings throughout study III, in the absence of Sln the myofibres of the mdx/SlnKO mice exhibited smaller CSA (P < 0.05) and a slow-to-fast fibre type transition (P < 0.05). Since promoting the slow-fibre population is considered a valid therapeutic strategy with the leading mechanistic hypothesis being an induction of utrophin, the slow-to-fast fibre type shift in the mdx/SlnKO mice is maladaptive. Indeed, this fibre type shift did result in lowered utrophin expression compared with mdx (P < 0.05). Utrophin is a dystrophin homolog that provides compensatory membrane stability in the absence of dystrophin, and its expression is largely controlled by calcineurin. Thus, these results are in support of SLN’s role in activating calcineurin and can account for the worsened dystrophic pathology with greater myofibre degeneration revealed by an elevated serum creatine kinase concentration (P < 0.05) and greater variation in the minimal Feret’s diameter (P < 0.05), an indicator of dystrophic pathology. The increased variability in fibre diameter is likely due to the combined effects of increased degeneration, reduced myofibre cross-sectional area, and impaired regenerative capacity (P < 0.05) in the absence of Sln.
Taken together the results from study III have answered the questioned pertaining to SLN’s role in skeletal muscle disease, a question that has remained elusive for many years. These findings clearly demonstrate that SLN is vital in activating muscle growth and remodeling and counters disease pathology by promoting calcineurin signaling. Combined with the findings in the PlnOE mouse in study I, the results from study III show that although PLN and SLN are structurally homologous and are both capable of inhibiting SERCA function, their physiological roles in skeletal muscle may differ. Specifically, increased PLN expression can cause skeletal muscle atrophy and disease whereas increased SLN is required to counter it. Thus, the collective results from this thesis reveal the importance of PLN and SLN in skeletal muscle disease.||en