Characterizing the Roles of Myoregulin and Dwarf Open Reading Frame in Regulating Calcium Homeostasis in Muscle

Abstract

The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are responsible for inducing muscle relaxation and are integral to the maintenance of intracellular calcium (Ca2+) homeostasis. As such, their activity is modulated by multiple regulatory proteins. Two well-characterized regulators of SERCAs are the homologous proteins sarcolipin (SLN) and phospholamban (PLN), which act to reduce the pump’s Ca2+ affinity/maximal activity. Though PLN and SLN share structural and functional homology, they exhibit distinct and diverse biological roles. It is well established that through their interaction with the SERCA pumps, both proteins are key regulators of muscle contraction/relaxation and both confer protection to the pump under times of cellular stress. However, SLN but not PLN, increases the amount of energy required by SERCA pumps, making SLN a key modulator of skeletal muscle energy metabolism, and protective against diet-induced obesity. Moreover, SLN plays a critical role in activating calcium signaling pathways that control adaptations in muscle mass and fiber type under conditions of muscle overload, disuse and disease. Myoregulin (MLN) and dwarf open reading frame (DWORF) are newly discovered protein regulators of SERCAs that share extensive sequence homology with SLN and PLN, and physically interact with the pump to regulate Ca2+-handling in muscle. Our understanding of these new regulators is still in its infancy. MLN has been shown to exert an inhibitory role similar to that of PLN and SLN by lowering SERCA Ca2+ affinity. Meanwhile, DWORF has been shown to act as a positive regulator of the pump both by removing the inhibition incurred by SLN, PLN and MLN and by directly stimulating SERCA Ca2+ affinity and/or maximal activity. While MLN and DWORF have emerged as key regulators of Ca2+ homeostasis and muscle contractility, it is unknown whether MLN and DWORF share similar roles to PLN and SLN and/or offer their own distinct and diverse physiological roles. It is well established that SERCAs are highly susceptible to damage and inactivation by high levels of oxidative stress and that their function is commonly impaired under conditions of muscle overload, disuse and disease. Thus, it is imperative to determine whether MLN and DWORF can protect the SERCA pumps from cellular stress. Furthermore, SLN’s ability to uncouple SERCA Ca2+ transport from ATP hydrolysis, and in doing so, alter SERCAs’ Ca2+ transport efficiency, is a vital mechanism underlying energy metabolism in skeletal muscle. Investigating whether MLN and DWORF can also contribute to muscle based adaptive thermogenesis by altering SERCA efficiency could offer new avenues for investigating whole body and muscle based metabolism. Finally, these four regulatory proteins appear to share overlap in their expression patterns, therefore, understanding whether the expression pattern of these two newly discovered proteins is altered by the absence of SLN and/or PLN would offer valuable insight into governance of intracellular Ca2+ homeostasis. To that end, the primary objectives for this thesis were to: 1) investigate whether the interaction between MLN:SERCA and DWORF:SERCA can protect the pump from damage during times of cellular stress, such as the thermal instability caused by heat shock; 2) determine whether MLN and/or DWORF were able to alter SERCA Ca2+ pumping efficiency; and 3) examine the potential compensatory changes in expression by evaluating MLN and DWORF gene and protein expression in Sln-/-, Pln-/-, and Sln-/-/Pln-/- mice. To address the first objective, HEK-293 cells were transfected for cDNA encoding SERCA1a or SERCA2a alone (20 ug) and co-transfected for cDNA encoding SERCA1a or SERCA2a (10 ug) at a molar ratio of 1:1 to those encoding MLN or DWORF (10 ug) with a total plasmid DNA of 20 µg. To induce heat stress (HS), prior to harvesting half of the cells from the groups were exposed to 40°C for one hour (HS) while the other half of the cells remained incubated at 37°C to be used as controls (CTL). Effects of HS on Ca2+-dependent SERCA activity was evaluated on crude cell homogenate over Ca2+ concentrations ranging from pCa 6.85 to 4.80 in the presence of the Ca2+ ionophore A23187 using a Ca2+-dependent, enzyme-linked spectrophotometric plate reader assay. Furthermore, effects of HS on the oxidation and nitrosylation of the SERCA protein were determined by western blotting on crude cell homogenate to assess oxidation by measuring protein carbonyls and nitrosylation by measuring nitrotyrosine formation. HS treatment of cells transfected with SERCA1a and SERCA2a alone significantly reduced maximal activity of both isoforms (p < 0.05) and resulted in stark elevations in both oxidation (p < 0.05) and nitrosylation (p < 0.05) of the pumps compared to CTL. In contrast, exposure of cells co-transfected with SERCA1a and MLN or DWORF and cells co-transfected with SERCA2a and MLN or DWORF to HS maintained maximal activity at levels resembling that of CTL (p > 0.05) and prevented SERCA oxidation (p > 0.05) and nitrosylation (p > 0.05), again keeping levels comparable to CTL. The results of this thesis are indicative of MLN and DWORF stabilizing SERCA structure and function during cellular stress by preserving maximal pump activity of both SERCA isoforms while minimizing oxidation and nitrosylation of the pump in the face of HS. To investigate the second objective, HEK-293 cells were transfected for cDNA encoding SERCA1a or SERCA2a alone (20 ug) and co-transfected for cDNA encoding SERCA1a or SERCA2a (10 ug) at a molar ratio of 1:1 to those encoding MLN or DWORF (10 ug) with a total plasmid DNA of 20 µg. Both Ca2+ uptake and Ca2+-ATPase activity were measured on crude cell homogenate prepared from the transfected cells. Ca2+-dependent SERCA activity was assessed over Ca2+ concentrations ranging from pCa 6.85 to 4.80 in presence and absence of the Ca2+ ionophore A23187 using a spectrophotometric plate reader assay. SERCA-mediated Ca2+ uptake was measured in the presence and absence of the precipitating anion, oxalate, using the fluorescent dye Indo-1 and spectrofluorometric plate reader assay. SERCA coupling ratio was calculated by dividing Ca2+ uptake by Ca2+-ATPase rates across different pCa values. The ionophore ratio was determined at each pCa to provides an additional way of assessing SERCA efficiency and was calculated by dividing SERCA activity measured in the presence of the ionophore by SERCA activity measured in the absence of the ionophore. In both the presence (p < 0.05) and absence of ionophore (p < 0.05), MLN significantly depressed the maximal rate of ATP consumption (Vmax) and SERCA’s Ca2+ affinity. Moreover, in both the presence (p < 0.05) and absence of oxalate (p < 0.05), MLN was shown to reduce SERCA Ca2+ uptake. However, MLN did not affect the Ca2+/ATP coupling ratio of SERCA1a and SERCA2a pumps at maximal (presence of ionophore/oxalate) and physiological (absence of ionophore/oxalate) conditions. Nor did MLN alter the ionophore ratio at each pCa. Thus, MLN does not have a bearing on SERCA pumping efficiency and is unlikely to contribute to muscle based energy expenditure. When examined in both the presence (p > 0.05) and absence (p > 0.05) of a Ca2+ gradient, DWORF had no bearing on the apparent affinity of SERCA1a and SERCA2a for Ca2+, no effect on maximal SERCA activity, no impact on the rate of SERCA-mediated Ca2+ uptake and did not alter the apparent coupling ratio or ionophore ratio, as no difference was observed between cells transfected with SERCA1a alone and those co-transfected with SERCA1a and DWORF or for cells transfected with SERCA2a alone and those co-transfected with SERCA2a and DWORF for any of these measures. Furthermore, no significant differences in Ca2+ affinity and maximal activity were observed in cells that were transfected with either SERCA isoform alone or co-transfected with DWORF (1:3) in either maximal (p > 0.05) or physiological (p > 0.05) conditions. Interestingly, DWORF was shown to increase SERCA apparent affinity for Ca2+, maximal SERCA activity and the rate of SERCA-mediated Ca2+ uptake in both the presence (p < 0.05) and absence (p < 0.05) of a Ca2+ gradient in cells co-transfected with SERCA1a or SERCA2a and DWORF at a molar ratio of 1:5. However, despite the activation of SERCA observed with a five-fold overexpression of DWORF, the apparent coupling ratio and ionophore ratio remained unaltered, therefore indicating that DWORF does not influence SERCA pumping efficiency. Lastly, to evaluate the third objective, western blotting was performed on soleus, left ventricle and atrial muscle isolated from 4-6-month-old male and female wild type mice and compared to single knockout (Sln-/- and Pln-/-) mice and double knockout (Sln-/-/Pln-/-) mice to examine if ablation of one or two SERCA regulatory protein(s) would result in compensatory changes in the expression of other SERCA regulatory proteins. In the Pln-/- genotype, no change in MLN or DWORF protein expression was detected with the ablation of PLN in both soleus (p > 0.05) and left ventricle (p > 0.05) muscle in either male nor female mice. Likewise, the Sln-/- genotype revealed no change in MLN or DWORF protein expression in the absence of SLN in both soleus (p > 0.05) and atrial (p > 0.05) muscle in either sex. Similarly, in the Sln-/-/Pln-/- model, the results for both proteins in both sexes matched what was observed in the single knockout models, as the ablation of both SLN and PLN did not result in any significant changes in the expression of MLN and DWORF (p > 0.05). To compliment the protein expression analysis, real-time PCR (qPCR) was performed on SOL and LV muscles from the same WT and knockout male and female mice to investigate potential changes in Mln and Dworf gene expression in the presence and absence of Sln and Pln expression individually and dual ablation. Gene expression of Mln was only altered in left ventricle tissue of female DKO, showing an elevation relative to WT (p < 0.05). The change in gene expression was generally unaltered in skeletal muscle tissue, with only soleus of male Sln-/- mice showing a significant elevation in Dworf expression (p < 0.05). In contrast, cardiac tissue exhibited a more complicated story: left ventricle tissue of male and female Pln-/- mice showed either a trending increase (p > 0.05) or significant increase (p < 0.05), respectively. Similarly, atria of Sln-/- male mice exhibited a trending increase in Dworf expression (p > 0.05), whereas, atria of Sln-/- female mice exhibited a significant decrease (p < 0.05). Finally, left ventricle and atrial tissue of both male and female Sln-/-/Pln-/- mice displayed a significant decline in Dworf expression (p < 0.05). Overall, this thesis has revealed that i) similar to SLN and PLN, MLN and DWORF stabilize SERCA structure and function during cellular stress; ii) like PLN, MLN negatively regulates SERCA kinetics without altering transport efficiency; iii) unlike any other known regulator, DWORF positively modulates the pump by enhancing SERCA apparent Ca2+ affinity, maximal activity and the rate of Ca2+ uptake without changing SERCA pumping efficiency; and iv) under basal conditions, the protein expression levels of MLN and DWORF are not altered by the presence/absence of either PLN and SLN alone or the presence/absence of both PLN and SLN together.