Effect of stress on the function of Na+/K+-ATPase and regulation of energy metabolism in skeletal muscleJan, Vid (Avtor) Pirkmajer, Sergej (Mentor) Podbregar, Matej (Komentor) Na+/K+-ATPaseFXYD1FXYD5AMPKskeletal musclehuman skeletal muscle cellsischaemiablood flow restriction trainingmetforminsulfasalazineAMPK activatorsde novo innervationdifferentiationAIMS: General purpose of this doctoral dissertation was to evaluate molecular mechanisms, which, during stressful conditions, alter Na+/K+-ATPase (NKA) activity and disturb regulation of energy metabolism in skeletal muscle. Specific aims were: (1) to establish an in vitro experimental model for the observation of NKA function and metabolism in skeletal muscle, in which physiological and biochemical characteristics would move closer to in vivo conditions compared to present experimental models; (2) to study molecular mechanisms through which hypoxia affects the functioning of NKA in skeletal muscle and (3) explore how metabolic changes affect NKA function in skeletal muscle. HYPOTHESES: We explored three hypotheses: (H1) Myotubes innervated in vitro and mature muscle fibres in vivo have a similar expression pattern of NKA and FXYD proteins. (H2) Hypoxia alters NKA function in skeletal muscle via activation of 5' AMP-activated protein kinase (AMPK). (H3) Modulation of NKA by pharmacological activators of AMPK depends on the metabolic state of skeletal muscle. METHODS: As an experimental model we used innervated and non-innervated human skeletal muscle cells (HSMC). We also used skeletal muscle biopsies of patients that were exposed to blood flow restriction (BFR) training. For analyses we used appropriate biomolecular methods (western-blot, RT-qPCR). RESULTS: Ad H1: Non-innervated HSMC are the most common cell model for skeletal muscle research. These cells predominantly express NKA?1 while skeletal muscles in vivo express higher amounts of NKA?2. Differentiation of HSMC increased the expression of NKA and FXYDs, which are more abundant in mature skeletal muscle, but innervation itself caused only minor additional changes. Ad H2: Knee injury patients are commonly exposed to hypoxia during surgery, which can also cause metabolic disorders or muscle damage. Energy stress activates AMPK, which has an important role in modulating energy metabolism. A study on skeletal muscle cells indicated that AMPK stimulation could lead to increased NKA activity, which is why we presumed that NKA function might increase in hypoxic conditions. We explored possible effects of low load training with blood flow restriction (LL-BFR training) of knee injury patients on NKA function in skeletal muscles. LL-BFR training increased NKA?1 content, which may have an important role in muscle hypertrophy. Increase in NKA?1 might lead to increased muscle mass of knee injury patients. We observed no changes in AMPK signalling. Ad H3: Increased activity of AMPK in skeletal muscles leads to improved energy status. Metformin, a known indirect AMPK activator, lowered mRNA expression of NKA only in ischaemic conditions, but not under normoxic or hypoxic conditions. We also evaluated sulfasalazine (SSZ), a salicylate derivative, for its potential effect on AMPK activity. SSZ increased AMPK activity in L6 cells and to minor extent in HSMC. CONCLUSIONS: (1) Using cell-culture homogenates we could not detect significant effect of innervation on the expression of NKA, FXYD1 and FXYD5. Our results therefore do not support the idea that innervated human myotubes in vitro display a similar expression pattern as myofibers in vivo (2) We showed that LL-BFR training increased the expression of NKA?1, which might lead to increased muscle mass, but our results do not provide evidence that hypoxia alters NKA function via AMPK (3) We showed that effects of metformin on NKA expression were dependent on energy status of skeletal muscle cells, which supports the third hypothesis. Also, SSZ, a known antirheumatic drug, increased AMPK activity in L6 cells and to a smaller extent in HSMC.20212021-06-20 07:15:47Doktorsko delo/naloga127697VisID: 16472COBISS_ID: 126742019sl