Effect of stress on the function of Na+/K+-ATPase and regulation of energy metabolism in skeletal muscle

Jan, Vid (avtor), Pirkmajer, Sergej (mentor), Podbregar, Matej (komentor)

AIMS: 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.

• Jezik: Angleški jezik
• Ključne besede: Na+/K+-ATPase, FXYD1, FXYD5, AMPK, skeletal muscle, human skeletal muscle cells, ischaemia, blood flow restriction training, metformin, sulfasalazine, AMPK activators, de novo innervation, differentiation
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2021
• PID: 20.500.12556/RUL-127697
• COBISS.SI-ID: 126742019
• Datum objave v RUL: 20.06.2021

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Vpliv stresnih razmer na delovanje Na+/K+-ATPaze in uravnavanje energijske presnove v skeletni mišici

Izvleček

CILJI: Glavni namen doktorske disertacije je bil proučiti molekularne mehanizme, ki v stresnih razmerah spremenijo delovanje Na+/K+-ATPaze in negativno vplivajo na uravnavanje energijske presnove v skeletni mišici. Specifični cilji so bili: (1) vzpostavitev in vitro celičnega modela za proučevanje delovanja NKA in presnove v skeletni mišici, ki bi bil po fizioloških in biokemijskih značilnostih skeletnomišičnih celic bolj podoben in vivo mišičnim vlaknom, kot trenutno uveljavljeni modeli, (2) proučiti molekularne mehanizme, ki preko hipoksije vplivajo na delovanje NKA v skeletni mišici in (3) raziskati, kako presnovne spremembe vplivajo na delovanje NKA v skeletni mišici. HIPOTEZE: Preverili smo tri hipoteze: (H1) Oživčene mišične cevčice v kulturi imajo podoben vzorec izražanja podenot NKA in proteinov FXYD kot zrela mišična vlakna. (H2) Hipoksija skeletne mišice vpliva na delovanje NKA prek aktivacije z AMP aktivirane protein-kinaze (AMPK). (H3) Farmakološki aktivatorji AMPK vplivajo na delovanje NKA v odvisnosti od presnovnega stanja skeletne mišice. METODE: Kot eksperimentalni model smo uporabili inervirane in neinervirane humane skeletne mišične celice (HSMC). Uporabili smo tudi mišične biopsije pacientov, ki so bili izpostavljeni ishemični vadbi. Za analizo smo uporabili primerne biomolekularne metode (odtis western in obratna transkripcija z verižno reakcijo s polimerazo - RT-qPCR). REZULTATI: Ad H1: Neinervirane cevčice so najpogosteje uporabljen celični model za proučevanje skeletne mišice. Te celice v večinskem deležu izražajo NKA α1/β-heterodimere, medtem ko mišična vlakna in vivo v največji meri izražajo α2/β-heterodimere. Diferenciacija HSMC je zvišala izražanje NKA2 in FXYD1, ki sta visoko zastopana v zreli skeletni mišici, inervacija pa ni imela signifikantnega vpliva na mRNA izražanje podenot NKA in FXYD proteinov. Ad H2: Pacienti s poškodbo kolena so med operacijo pogosto izpostavljeni hipoksiji, kar lahko vodi v presnovne motnje in poškodbo mišic. Energijski stres aktivira AMPK, ki ima pomembno vlogo pri uravnavanju energijske presnove. Študija na skeletno-mišičnih celicah je pokazala, da lahko stimulacija AMPK aktivira NKA, zaradi česar smo sklepali, da bi se NKA v skeletnih mišicah v hipoksiji lahko aktivirala. Proučili smo morebitne učinke ishemične vadbe proti majhnemu uporu (LL-BFR) pri pacientih s poškodbo kolena na delovanje NKA v skeletni mišici. LL-BFR vadba je povečala vsebnost NKAα1, kar bi lahko igralo vlogo pri trofičnih učinkih LL-BFR vadbe pri pacientih s poškodbo kolena. Sprememb v signalizaciji AMPK nismo zaznali. Ad H3: Povečana aktivnost AMPK v skeletni mišici vodi v izboljšanje presnovnega stanja. Metformin, znan posredni aktivator AMPK, je znižal mRNA izražanje podenot NKA le v ishemičnih razmerah. Pokazali smo tudi, da sulfasalazin (SSZ), derivat salicilata, lahko aktivira AMPK v L6 podganji skeletnomišični celični liniji in v manjši meri v HSMC. ZAKLJUČKI: (1) V homogenatih celičnih kultur nismo zaznali statistično značilnih učinkov inervacije na izražanje podenot NKA, FXYD1 in FXYD5. Naši rezultati ne podpirajo hipoteze, da imajo in vitro inervirane HSMC podoben profil izražanja kot mišična vlakna in vivo. (2) Pokazali smo, da LL-BFR vadba poviša izražanje NKAα1, kar bi lahko vodilo v povečanje mišične mase, ne moremo pa potrditi, da hipoksija spremeni delovanje NKA preko AMPK (3) Pokazali smo, da so učinki metformina na izražanje NKA odvisni od presnovnega stanja skeletnomišičnih celic, kar potrjuje tretjo hipotezo. Poleg tega je sulfasalazin, znan antirevmatik, povišal aktivnost AMPK v L6 celicah in v manjši meri tudi v HSMC.

• Ključne besede: Na+/K+-ATPaza, FXYD1, FXYD5, skeletna mišica, humane skeletne mišične celice, ishemia, ishemična vadba proti majhnemu uporu, metformin, sulfasalazine, aktivatorji AMPK, de novo inervacija, diferenciacija