Assessment of Metabolic Therapy for Acute Heart Failure

Kimar, Charlene Patricia (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Introduction Acute heart failure (AHF) is the most common primary diagnosis for hospitalized heart disease cases in Africa. Increased fatty acid oxidation (FAO) with heart failure (HF) triggers detrimental effects on the myocardium, we hypothesized that diabetic rat hearts subjected to AHF display lower cardiac function vs. controls and that Trimetazidine (TMZ) (a partial FAO inhibitor) counters this effect. Aims 1)To establish an ex vivo AHF model for diabetic hearts; 2) Assess whether TMZ treatmentoffers cardioprotection to diabetic rat hearts subjected to an AHF protocol; and 3) Delineate underlying mechanisms by evaluating markers for oxidative stress, mitochondrial uncoupling, apoptosis and metabolic dysregulation. Methods Vehicle control male Wistar rats were injected with citrate buffer. To induce diabetes rats were administered streptozotocin (60 mg/kg) for one week vs. non-diabetic controls. Hearts were perfused on the Langendorff retrograde perfusion system for three phases: Stabilization - (11 mM glucose- non-diabetic, and 30 mM glucose- diabetic hearts) at 100 cm H2O (30 min); AHF – (1.5 mM palmitic acid, 2.5 mM glucose) at 20 cm H2O (35 min); and Recovery– (1.5 mM palmitic acid, 11 mM glucose or 30 mM glucose) at 100 cm H2O (30 min). 1 μM TMZ was administered at the start of recovery. In addition, we evaluated necrosis and infarct size by tetrazolium (TTC) staining at the end of the AHF phase. Western blotting was performed for markers of apoptosis (pBAD/BAD), oxidative stress (superoxide dismutase 2 [SOD2], conjugated dienes [CDs], thiobarbituric acid reactive substances (TBARS), reduced/oxidized glutathione [GSH/GSSG] analysis, oxygen radical absorbance capacity [ORAC]), mitochondrial uncoupling (uncoupling protein 2 [UCP2]) and metabolic dysregulation (advanced glycation end product [AGE] and polyol pathway analyses). We investigated direct effects of TMZ (1 μM) in H9c2 cardiomyoblasts exposed to 500 μM palmitate for 21 hours and assessed the effects of TMZ treatment on fatty acid-induced oxidative stress and apoptosis. Results Reduced function was seen for all groups in recovery vs. controls, while AHF-diabetic showed worse outcomes vs. AHF alone. TMZ treatment resulted in a robust increase in left ventricular developed pressure (LVDP) for diabetic hearts vs. controls. Infarct size assessment showed no differences. TMZ treated diabetic hearts also displayed lower AGE and higher polyol pathway activation vs. respective controls. However, several markers of the AGE pathway did not show any significant differences for any groups. Non-diabetic and diabetic hearts displayed increased oxidative stress (TBARS) compared to their counterparts. TMZ treatment resulted in anti-apoptotic effects in hearts subjected to AHF. TMZ exhibited antioxidant effects by lowering fatty acid-induced mitochondrial oxidative stress in cells. Conclusion This study successfully established a novel ex vivo model of AHF for the diabetic rat heart, and TMZ treatment resulted in cardioprotection for diabetic hearts. Our data suggest that TMZ may mediate some of its cardioprotective effects by acting as an anti-oxidant to lower myocardial oxidative stress triggered during AHF. The findings also indicate that TMZ treatment may lower the formation of damaging AGEs in the diabetic heart. TMZ therefore, emerges as a putative therapeutic target to be considered as sole and/or combined treatment (with more conventional drugs) for AHF patients.

AFRIKAANSE OPSOMMING: Inleiding Akute hartversaking (AHV) is die mees algemene primêre diagnose vir gehospitaliseerde hartsiekte gevalle in Afrika. Verhoogde vetsuuroksidasie (VSO) met hartversaking (HV) veroorsaak skadelike effekte op die miokardium. Ons hipotiseer dat diabetiese rotharte blootgestel aan AHV verlaagde hartfunksie vertoon vs. kontroles en dat Trimetazidien (TMZ) (‘n gedeeltelike VSO inhibitor), hierdie effek teenwerk. Doelwitte 1) Om ‘n ex vivo AHV model vir diabetiese harte te vestig; 2) Assesseer of TMZ behandeling kardiobeskerming verleen aan diabetiese rotharte blootgestel aan ‘n AHV protokol; en 3) Karakterisering van onderliggende meganismes deur merkers vir oksidatiewe stres, mitochondriese ontkoppeling, apoptose en metaboliese wanregulering te ondersoek. Metodes Draer kontrole Wistar rot mannetjies was met sitraat buffer ingespuit. Om diabetes te induseer was rotte met streptozotosien (60 mg/kg) vir een week toegedien vs. nie-diabetiese kontroles. Harte is geperfuseer op die Langendorff retrograad perfusie sisteem vir drie fases: Stabilisasie – (11 mM glukose-nie-diabeties, en 30 mM glukose-diabetiese harte) teen 100 cm H2O (30 min); AHV – (1.5 mM palmitiensuur, 2.5 mM glukose) teen 20 cm H2O (35 min); en Herstel – (1.5 mM palmitiensuur, 11 mM glukose of 30 mM glukose) teen 100 cm H2O (30 min). 1 μM TMZ is aan die begin van herstel toegedien. Ons het addisioneel nekrose en infarktgrootte aan die einde van die AHV fase geevalueer met tetrazolium (TTC) kleuring. Western klad-analise is uitgevoer vir merkers van apoptose (pBAD/BAD), oksidatiewe stres (superoksieddismutase 2 [SOD2], gekonjugeerde diëne [CDs], tiobarbituursuur reaktiewe stowwe (TBARS), gereduseerde/geoksideerde glutatioon [GSH/GSSG] analise, suurstof radikaal absorbansie kapasiteit [ORAC]), mitochondriese ontkoppeling (ontkoppelingproteïen 2 [UCP2]) en metaboliese wanregulering (gevorderde glukasie eindproduk [AGE] en poliolpad analise). Ons het die direkte effek van TMZ (1 μM) in H9c2 kardiomioblaste, blootgestel aan 500 μM palmitaat vir 21 ure, ondersoek en die effek van TMZ behandeling op vetsuur-geïnduseerde oksidatiewe stres en apoptose, geassesseer. Resultate Verminderde funksie is waargeneem vir alle groepe in herstel vs. kontrole, terwyl AHV-diabete slegter uitkomste getoon het vs. slegs AHV. TMZ behandeling het gelei tot ‘n sterk toename in linker ventrikulêre ontwikkelde druk (LVDP) vir diabetiese harte vs. kontroles. Infarktgrootte assessering het geen verskille getoon nie. TMZ behandelde diabetiese harte het ook laer AGE en hoër poliolpad aktivering vs. onderskeidelike kontroles, getoon. ‘n Aantal merkers van die AGE pad het egter nie betekenisvolle verskille vir enige groepe gedemonstreer nie. Nie-diabetiese en diabetiese harte het verhoogde oksidatiewe stres (TBARS) in vergelyking met hul ekwivalente getoon. TMZ behandeling het anti-apoptotiese effekte teweeggebring in harte blootgestel aan AHV. TMZ het anti-oksidant effekte getoon deur die verlaging van vetsuur-geïnduseerde mitochondriese oksidatiewe stres in selle. Gevolgtrekking Hierdie studie het suksesvol ‘n nuwe ex vivo model vir AHV vir die diabetiese rothart gevestig, en TMZ behandeling het gelei tot kardiobeskerming vir diabetiese harte. Ons data stel voor dat TMZ die kardiobeskermende effekte daarvan medieer deur op te tree as anti-oksidant om miokardiale oksidatiewe stres veroorsaak tydens AHV, te verlaag. Die bevindinge dui ook aan dat TMZ behandeling die vorming van skadelike AGE in die diabetiese hart mag verlaag. TMZ verskyn dus as ‘n putatiewe terapeutiese teiken om in ag te neem as enkel en/of gekombineerde behandeling (met meer konvensionele geneesmiddels) vir AHV pasiënte.

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