An evaluation of trimetazidine as a therapeutic intervention in a newly established ex vivo mouse model of acute heart failure

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Introduction Acute heart failure (AHF) is the most common primary diagnosis for hospitalized heart diseases in Africa. Although Sub-Saharan women are more prone to suffer from de novo AHF at a much younger age, females have historically been underrepresented in biomedical research studies. As increased fatty acid oxidation (FAO) with heart failure triggers detrimental effects within the myocardium, we hypothesized trimetazidine (TMZ) (a partial FAO inhibitor) treatment will provide cardio protection to control and diabetic mouse hearts subjected to AHF. We further hypothesized that TMZ efficacy will be influenced by different phases of the estrous cycle. Aims 1) Establish an unique ex vivo AHF model using hearts isolated from db/db mice and their lean control littermates (db/+); 2) Evaluate whether FA-albumin filtering can replace the gold standard method of dialysis for perfusate preparation; 3) Assess whether we can identify the different phases namely proestrus, estrus (follicular phase), metestrus and diestrus (luteal phase) of the estrous cycle in the female mice; and 4) Evaluate TMZ as a therapeutic option in our ex vivo AHF model for normal and obese/diabetic mice, respectively, ascertain if there are sex-based differences, and determine whether the phases of the estrous cycle can influence cardio protection. Methods The Langendorff retrograde isolated heart perfusion system was employed to establish an ex vivo AHF model that consisted of three phases: Stabilization – Krebs-Henseleit buffer (10 mM glucose) at 100 cmH2O (25 minutes); Critical Acute Heart Failure (CAHF) – (2.5 mM glucose, 1.2 mM palmitic acid bound to 3% bovine serum albumin [BSA]) at 20 cmH2O (25 minutes); and Recovery Acute Heart Failure (RAHF) – (10 mM glucose, 1.2 mM palmitic acid bound to 3% BSA) at 100 cmH2O (25 minutes). 5 μM TMZ was administered in the perfusate at either the CAHF or RAHF phase for the full duration of the respective phase. The filter versus dialysis experiments were run for 30 minutes in Krebs-Henseleit buffer (10 mM glucose, 1.2 mM palmitic acid bound to 3% BSA). Phases of the estrous cycle were determined by vaginal smear cytology or “wet smears” and viewed under a light microscope. Enzyme-linked immunosorbent assays (ELISA) were utilized to measure serum hormonal levels while Western blotting was employed to assess protein expression levels. Results Our model mimicked de novo AHF in the switch from stabilization to CAHF and partial recovery in the switch from CAHF to RAHF. This study established that the dialysis method for FA-BSA preparations can be substituted by a simple filtering protocol. While vaginal smear cytology confirmed acyclicity of obese females (therefore lost follicular phase), lean females exhibited normal estrous cycle phases. Commercial ELISA kits were not adequately sensitive to detect hormonal fluctuations. All groups displayed a severe decrease in function during CAHF and recovery with RAHF (vs. CAHF). Lean and obese males benefited equally from TMZ treatment administered during the RAHF phase. The lean females in the two main phases of the estrous cycle (follicular and luteal) responded in distinct ways. Here lean follicular females were the only group to respond to TMZ treatment during the CAHF phase, while lean luteal females did not respond to therapy but rather displayed an inherent cardio protection that was lost with obesity. Obese luteal females also benefited from TMZ treatment during RAHF. No changes were observed in protein expression levels of 3-keotacyl-CoA thiolase (3-KAT) nor pyruvate dehydrogenase (PDH). Conclusion A novel ex vivo mouse AHF model has successfully been established and utilized the filtering method as opposed to the gold standard dialysis method. TMZ as a therapy for AHF showed great promise in improving functional recovery of mice subjected to the AHF protocol. Sex differences were present only in lean groups where the phases of the estrous cycle influenced therapy, while obesity only affected TMZ efficacy in females. The optimization of cardiac metabolism by TMZ emerges as a novel and worthy therapeutic option to investigate for the treatment of AHF in normal and diabetic patients (for both genders).AFRIKAANSE OPSOMMING: Inleiding Akute hartversaking (AHF) is die mees algemene primêre diagnose vir gehospitaliseerde hartsiektes in Afrika. Alhoewel vrouens van Sub-Sahara Afrika meer geneig is om te ly aan de novo AHF op 'n veel jonger ouderdom, is vrouens histories onderverteenwoordig in biomediese navorsingstudies. Verhoogde vetsuuroksidasie (FAO) tydens hartversaking lei tot nadelige gevolge binne die miokardium. Ons vermoed behandeling met trimetazidine (TMZ) ('n gedeeltelike FAO inhibitor) sal beskerming bied aan kontrole en diabeet muis harte onderworpe aan AHF. Ons vermoed verder dat TMZ se doeltreffendheid beïnvloed sal word deur verskillende fases van die estrussiklus. Doelwitte 1) Stel 'n unieke ex vivo AHF model op met behulp van harte geïsoleer vanaf db/db muise en hul skraal kontrole werpselmaats (db/+); 2) Evalueer of die filtrering van vetsuur-albumien die standaard metode van dialise vir perfusaat voorbereiding kan vervang; 3) Assesseer of ons die verskillende fases naamlik proestrus, estrus (follikulêre fase), metestrus en diestrus (luteale fase) van die estrussiklus in die vroulike muise kan identifiseer; en 4) Evalueer TMZ as 'n terapeutiese opsie in ons ex vivo AHF model vir normale en oorgewig/diabeet muise, onderskeidelik, stel vas of daar geslag-gebaseerde verskille teenwoordig is, en bepaal of die fases van die estrussiklus kardiale beskerming kan beïnvloed. Metodes Die Langendorff retrograde geïsoleerde hartperfusie stelsel is gebruik om 'n ex vivo AHF model te vestig wat bestaan uit drie fases: Stabilisasie – Krebs-Henseleit buffer (10 mM glukose) by 100 cmH2O (25 minute); Kritieke Akute Hartversaking (CAHF) – (2,5 mM glukose, 1,2 mM palmitiensuur gebind aan 3% van bees serum albumien [BSA]) by 20 cmH2O (25 minute); en Herstellende Akute Hartversaking (RAHF) - (10 mM glukose, 1,2 mM palmitiensuur gebind tot 3% BSA) by 100 cmH2O (25 minute). TMZ (5 μM) is toegedien in die perfusate óf by die CAHF of RAHF fase vir die volle duur van die onderskeie fase. Die filtrering teenoor dialise eksperimente is uitgevoer vir 30 minute met Krebs-Henseleit buffer (10 mM glukose, 1,2 mM palmitiensuur gebind tot 3% BSA). Fases van die estrussiklus was bepaal deur vaginale smeer sitologie of "nat smere" en besigtiging onder 'n ligmikroskoop. Ensiem-gekoppelde immunosorberende toetse (ELISAs) is gebruik om hormonale vlakke in serum te meet, terwyl Westerse klad analises gebruik is om vlakke van proteïen uitdrukking te evalueer. Resultate Ons model boots de novo AHF na in die oorskakeling van die stabilisering fase na CAHF en gedeeltelike herstelling in die oorskakeling van CAHF na RAHF. Hierdie studie het vasgestel dat die dialise metode vir die vetsuur-BSA voorbereidings vervang kan word deur 'n eenvoudige filtrasie protokol. Vaginale smeer sitologie het bevestig dat oorgewig wyfies nie normaal deur die estrussiklus sirkuleer nie, terwyl skraal wyfies wel normaal deur die fases van die siklus beweeg. Kommersiële ELISA toetse se sensitiwiteit was nie voldoende om hormonale afwykings vas te stel nie. Alle groepe het 'n ernstige afname in funksionaliteit getoon tydens CAHF en herstel met RAHF (teenoor CAHF). Skraal en oorgewig mannetjies het ewe veel voordeel getrek uit TMZ behandeling, toegedien tydens die RAHF fase. Die skraal wyfies in die twee hoof-fases van die estrussiklus (follikulêre en luteale fases) het op verskillende maniere gereageer. Hier was die skraal follikulêre wyfies die enigste groep om te reageer op die TMZ behandeling tydens die CAHF fase, terwyl skraal luteale wyfies nie reageer het op behandeling nie, maar eerder 'n inherente kardiale beskerming getoon het wat afwesig was met vetsug. Oorgewig luteale wyfies het ook voordeel getrek uit TMZ behandeling tydens RAHF. Geen veranderinge is waargeneem in proteïen uitdrukking vlakke van 3-keotacyl-KoA thiolase (3-KAT) en piruvaatdehidrogenasekompleks (PDH) nie. Slot 'n Nuwe ex vivo muis AHF model is suksesvol gevestig en die filtrasie metode was ingestel in teenstelling met die goue standaard dialise metode. As terapie teen AHF, is TMZ heel belowend in die verbetering van funksionele herstel van muise onderworpe aan die AHF protokol. Geslagsverskille is slegs waargeneem in skraal groepe waar die fases van die estrussiklus terapie beïnvloed het, terwyl TMZ doeltreffendheid slegs in wyfies deur vetsug affekteer is. Die optimisering van hart metabolisme deur TMZ terapie kom na vore as 'n nuwe en waardige terapeutiese opsie om te ondersoek vir behandeling van AHF in normale en diabetiese pasiënte (van beide geslagte)