7 research outputs found
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Human Tissue Engineered Model of Myocardial Ischemia-Reperfusion Injury
Timely reperfusion after a myocardial infarction is necessary to salvage the ischemic region; however, reperfusion itself is a major contributor to the final tissue damage. Currently, there is no clinically relevant therapy available to reduce ischemia-reperfusion injury. While many drugs have shown promise in reducing ischemia-reperfusion injury in preclinical studies, none of these drugs have demonstrated benefit in large clinical trials. Part of the failure to translate therapies can be attributed to the reliance on small animal models for preclinical studies. While animal models encapsulate the complexity of the systemic in vivo environment, they do not fully recapitulate human cardiac physiology.
In this thesis, we utilized cardiac tissue engineering methods in conjunction with cardiomyocytes derived from human induced pluripotent stem cells, to establish a biomimetic human tissue-engineered model of ischemia-reperfusion injury. The resulting cardiac constructs were subjected to simulated ischemia or ischemia-reperfusion injury in vitro. We demonstrated that the presence of reperfusion injury can be detected and distinguished from ischemic injury. Furthermore, we demonstrated that we were able to detect changes in reperfusion injury in our model following ischemic preconditioning, modification of reperfusion conditions, and addition of cardioprotective therapeutics. This work establishes the utility of the human tissue model in studying ischemia-reperfusion injury and the potential of the human tissue platform to help translate therapeutic strategies into the clinical setting
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The role of the large conductance Ca2+-activated K+ channel in adenosine receptor-mediated cytoprotection
The rat embryonic cardiomyoblast-derived H9c2 cell line is increasingly used for studies into cardioprotection, as these cells display similar properties to primary cardiomyocytes. Adenosine receptors are well known mediators of cardioprotection and trigger effectors such as the mitochondrial KATP channel – however, the role of the mitochondrial BKCa channel in adenosine receptor-mediated cardioprotection has not been investigated. GPCR assays provided evidence for functional expression of Gi-coupled adenosine A1 and κ-opioid receptors, Gs-coupled β2 adrenergic receptors and Gq-coupled UTP-binding P2Y purinergic receptors on H9c2 cells. Activation of the adenosine A1 receptor with CPA (N(6)-cyclopentyladenosine) provided significant protection against hypoxia-induced cell death in these cells, as did opening of a BKCa channel with NS1619. The location of this BKCa channel was confirmed to be the mitochondria by the probing of subcellular fractions with BKCa-specific antibodies. Interestingly, CPA-induced protection against hypoxia was blocked by inhibition of the BKCa channel
Efectos electrofisiológicos miocárdicos intrÃnsecos producidos por la inhibición del sistema de intercambio sodio-protón, en condiciones de normoxia, y tras la isquemia miocárdica regional. Estudio experimental.
RESUMEN
Hemos investigado: 1) los efectos protectores del intercambiador sodio-protón (NHE), con el 5-(N-etil-N-isopropil) amiloride (EIPA), a concentración 0.4 M, sobre la inducibilidad de la fibrilación ventricular (FV), y sobre la refractariedad miocárdica ventricular, tras la oclusión arterial coronaria; 2) los efectos electrofisiológicos intrÃnsecos producidos por la inhibición del NHE, administrando EIPA 0.4 y 1 M, en condiciones de normal oxigenación, sobre el automatismo sinusal, la conducción sinoauricular y auriculoventricular (AV), y sobre la refractariedad auricular, ventricular, y del sistema de conducción AV; y 3) los efectos electrofisiológicos miocárdicos intrÃnsecos producidos por la inhibición del NHE, con EIPA 0.4 y 1 M, sobre la frecuencia dominante (FD) de la FV inducida, en condiciones de normoxia, como caracterÃstica electrofisiológica estrechamente relacionada con la refractariedad ventricular.
Todos los experimentos se realizaron en corazón aislado y perfundido de conejo (preparación tipo Langendorff). Para conseguir el primer objetivo aplicamos el test del extraestÃmulo (TEE) ventricular con uno, dos, y tres extraestÃmulos, para tratar de inducir la FV. La aplicación del primer extraestÃmulo sirvió además para establecer el periodo refractario objeto de investigación. Las determinaciones se realizaron previamente y tras la oclusión arterial coronaria circunfleja, en dos grupos de experimentos: control y tratados con EIPA 0.4 M.
Para cumplimentar el segundo objetivo analizamos:
a) periodos refractarios efectivo y funcional auricular y del sistema de conducción AV mediante el TEE auricular, periodos refractarios efectivo y funcional ventricular mediante el TEE ventricular; b) automatismo sinusal mediante la determinación de la longitud del ciclo sinusal y del tiempo de recuperación del nodo sinusal; y c) tiempo de conducción sinoauricular con el test de Narula, conducción AV y conducción ventriculoauricular retrógrada, mediante la medición de la longitud del ciclo de Wenckebach y la longitud del ciclo de Wenckebach retrógrado respectivamente. Finalmente, para acometer el tercer objetivo analizamos, por el método de Welch, la FD media, máxima y mÃnima de la FV inducida por estimulación a frecuencias crecientes. Los parámetros citados se estudiaron en situación control e inhibiendo el NHE, con EIPA. Utilizamos dos concentraciones de dicho inhibidor: 0.4 y 1 M; la primera se halla en el rango de la IC50 para el NHE y la segunda es la concentración más comúnmente usada en los estudios de protección miocárdica.
La inhibición del NHE con EIPA 0.4 M protegió al miocardio isquémico, al dificultar la inducibilidad de la FV. Este efecto protector, probablemente está relacionado, al menos en parte, con el mantenimiento de la refractariedad ventricular en el miocardio isquémico, lo cual ha sido también observado en el presente trabajo. Excepto para el caso del periodo refractario efectivo del sistema de conducción AV, que aumentó ligeramente con EIPA 1 M, la inhibición del NHE con EIPA 0.4 y 1 M en condiciones de normal oxigenación, no modificó la refractariedad intrÃnseca ventricular, auricular, ni del sistema de conducción AV, asà como tampoco la FD de la FV inducida, mientras que deprimió ligeramente el cronotropismo sinusal y la conducción AV, si bien estos últimos podrÃan ser efectos secundarios, relacionados con la capacidad depresora del automatismo sinusal y la conducción AV que ejerce el amiloride, precursor del EIPA.
El hecho de que la inhibición del NHE, manifestara efectos electrofisiológicos antiarrÃtmicos intrÃnsecos sobre el miocardio isquémico, impidiendo la caÃda de la refractariedad, y no ejerciera ninguna modificación electrofisiológica, en condiciones de normal oxigenación, sobre la refractariedad y la FD de la FV, propiedades ligadas a la aparición de arritmias por reentrada, coincide con lo relatado por diversos investigadores acerca del escaso, sino nulo, papel del NHE en situación fisiológica y su importancia en circunstancias fisiopatológicas como la isquemia.
__________________________________________________________________________________________________We have investigated the protective effects of the sodium-proton exchanger (NHE) inhibition using 5-(N-ethyl-N-isopropyl) amiloride (EIPA) 0.4 M, on the incidence of induced ventricular fibrillation (VF), and on intrinsic ventricular refractoriness, after circumflex coronary artery occlusion. Experiments were performed on isolated rabbit heart. To induce VF extrastimulus test with one, two and three extrastimuli, was used; the first extrastimulus application also served to determine refractory periods. Parameters were determined previously and after coronary occlusion, in both control and EIPA 0.4 M treated groups. In order to investigate whether the NHE inhibition produces some myocardial intrinsic electrophysiological effect, we have also investigated using EIPA, 0.4 and 1 M, in normoxic condition: a) the sinus cycle length and sinus node recovery time, as an automatism index; b) the sinoatrial conduction (Narulas test), and atrioventricular (AV) and ventricleatrial conduction, by anterograde and retrograde Wenckebach cycle length determination respectively; c) atrial, ventricular, and AV conduction system, effective and functional refractory periods using the extrastimulus test; and d) mean, minimum and maximum dominant frequency (DF) of VF by the Welchs method, which are properties closely related with ventricular refractoriness.
NHE inhibition with 0.4 M protected the ischemic myocardium, decreasing VF inducibility, at least in part, by the maintenance of ischemic myocardium refractoriness, observed in this study. No modification on refractoriness was observed, in normoxic conditions, except an AV conduction system effective refractory period increase, with EIPA 1 M. The DF of VF was either not modified. EIPA 0.4 and 1 M also slightly depressed sinus chronotropism and AV conduction
The effect of creatine supplementation on myocardial metabolism and function in sedentary and exercised rats
Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Background: There has been a dramatic increase in the use of dietary creatine
supplementation among sports men and women, and by clinicians as a therapeutic
agent in muscular and neurological diseases. The effects of creatine have been studied
extensively in skeletal muscle, but knowledge of its myocardial effects is limited.
Objectives: To investigate the effects of dietary creatine supplementation with and
without exercise on 1) basal cardiac function, 2) susceptibility to ischaemia/reperfusion
injury and 3) myocardial protein expression and phosphorylation and 4) mitochondrial
oxidative function.
Methods: Male Wistar rats were randomly divided into control or creatine supplemented
groups. Half of each group was exercise trained by swimming for a period of 8 weeks, 5
days per week. At the end of the 8 weeks the open field test was performed and blood
corticosterone levels were measured by RIA to determine whether the swim training
protocol had any effects on stress levels of the rats. Afterwards hearts were excised and
either freeze-clamped for biochemical and molecular analysis or perfused on the
isolated heart perfusion system to assess function and tolerance to ischaemia and
reperfusion. Five series of experiments were performed: (i) Mechanical function was
documented before and after 20 minutes global ischaemia using the work heart model,
(ii) A H2O filled balloon connected to a pressure transducer was inserted into the left
ventricle to measure LVDP and ischaemic contracture in the Langendorff model, (iii)
The left coronary artery was ligated for 35 minutes and infarct size determined after 30
minutes of reperfusion by conventional TTC staining methods. (iv) Mitochondrial
oxidative capacity was quantified. (v) High pressure liquid chromatography (HPLC) and
Western Blot analysis were performed on blood and heart tissue for determination of
high energy phosphates and protein expression and phosphorylation.
Results: Neither the behavioural studies nor the corticosterone levels showed any
evidence of stress in the groups investigated. Hearts from creatine supplemented
sedentary (33.5 ± 4.5%), creatine supplemented exercised rats (18.22 ± 6.2%) as well
as control exercised rats (26.1 ± 5.9%) had poorer aortic output recoveries than the
sedentary control group (55.9 ± 4.35% p < 0.01) and there was also greater ischaemic
contracture in the creatine supplemented exercised group compared to the sedentary
control group (10.4 ± 4.23 mmHg vs 31.63 ± 4.74 mmHg). There were no differences in
either infarct size or in mitochondrial oxygen consumption between the groups. HPLC
analysis revealed elevated phosphocreatine content (44.51 ±14.65 vs 8.19 ±4.93
nmol/gram wet weight, p < 0.05) as well as elevated ATP levels (781.1 ±58.82 vs 482.1
±75.86 nmol/gram wet weight, p<0.05) in blood from creatine supplemented vs control
sedentary rats. These high energy phosphate elevations were not evident in heart
tissue and creatine tranporter expression was not altered by creatine supplementation.
GLUT4 and phosphorylated AMPK and PKB/Akt were all significantly higher in the
creatine supplemented exercised hearts compared to the control sedentary hearts.
Conclusion: This study suggests that creatine supplementation has no effects on basal
cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise
trained animals by increasing the ischaemic contracture and decreasing reperfusion
aortic output. Exercise training alone also significantly decreased aortic output recovery.
However, the exact mechanisms for these adverse myocardial effects are unknown and
need further investigation.AFRIKAANSE OPSOMMING: Agtergrond: Die gebruik van kreatien as dieetaanvulling het in die afgelope aantal jaar
dramaties toegeneem onder sportlui, sowel as mediese praktisyns wat dit as ‘n
terapeutiese middel vir die behandeling van spier- en neurologiese siektes aanwend.
Die effekte van kreatien op skeletspier is reeds deeglik ondersoek, maar inligting
aangaande die miokardiale effekte van die preperaat is beperk.
Doelwitte: Om die effekte van kreatien dieetaanvulling met of sonder oefening ten
opsigte van die volgende aspekte te ondersoek: 1) basislyn miokardiale funksie, 2)
vatbaarheid vir iskemie/herperfusie besering, 3) proteïenuitdrukking en -fosforilering in
die miokardium en 4) mitochondriale oksidatiewe funksie.
Metodes: Manlike Wistar rotte is ewekansig in kontrole of kreatien aanvullings groepe
verdeel. Helfte van elke groep is aan oefening in die vorm van swemsessies, vir ‘n
periode van 8 weke, 5 dae per week blootgestel. Gedrags- en biochemiese toetse is
aangewend om die moontlike effek van die swemprotokol op die rotte se stres vlakke te
bepaal. In hierdie verband is die oop area toets gebruik, asook bloed kortikosteroon
vlakke gemeet deur radioaktiewe immuunessais. Harte is daarna uit die rotte
gedissekteer en gevriesklamp vir biochemiese en molekulêre analise, of geperfuseer op
die geïsoleerde werkhart perfusiesisteem om sodoende funksie en weerstand teen
iskemie en herperfusie beskadeging te bepaal. Vyf eksperimentele reekse is uitgevoer:
(i) Meganiese funksie is noteer voor en na 20 minute globale isgemie in die werkhart
model; (ii) ‘n Water gevulde plastiek ballon, gekoppel aan ‘n druk omsetter, is in die
linker ventrikel geplaas om sodoende linker ventrikulêre ontwikkelde druk (LVDP),
asook iskemiese kontraktuur te meet; (iii) Linker koronêre arterie afbinding is vir ‘n
periode van 35 minute toegepas en die infarktgrootte bepaal na 30 minute herperfusie
deur gebruik te maak van standaard kleuringsmetodes; (iv) Mitochondriale oksidatiewe
kapasiteit is gemeet; (v) Hoë druk vloeistof chromatografie (HPLC) en Western Blot
analises is uitgevoer op bloed en hartweefsel vir die bepaling van hoë energie fosfate
(HEFe), sowel as proteïenuitdrukking en -fosforilering.
Resultate: Beide gedragsstudies en kortikosteroonvlakke het geen teken van stres in
die betrokke groepe getoon nie. Die groep blootgestel aan kreatienaanvulling en
oefening se harte het na iskemie funksioneel swakker herstel as harte van die
onaktiewe kontrole groep (18.22±6.2% vs 55.9±4.35%; p<0.01), asook ‘n groter
ikgemiese kontraktuur in vergelyking met die onaktiewe kontrole groep ontwikkel
(31.63±4.74 mmHg vs 10.4±4.23 mmHg). Daar was geen verskille in infarktgrootte of
mitochondriale suurstofverbruik tussen die verskillende groepe waargeneem nie. HPLC
analise het verhoogde fosfokreatien (44.51±14.65 vs 8.19±4.93 nmol/gram nat gewig,
p<0.05) en adenosientrifosfaat (ATP) bloedvlakke (781.1±58.82 vs 482.1±75.86
nmol/gram nat gewig, p<0.05) in kreatien aanvullings vergelyk met die kontrole groepe
getoon. Daar was egter geen meetbare veranderings in HEF vlakke in hartweefsel nie.
Gepaardgaande hiermee het kreatienaanvulling geen effek gehad op die uitdrukking va
die kreatien transporter nie. In vergelyking met onaktiewe kontrole harte was GLUT4, en
fosforileerde AMPK en PKB/ Akt beduidend hoër in harte van geoefende rotte met
kreatienaangevulling.
Gevolgtrekking: Hierdie data dui daarop dat kreatienaanvulling geen effek op basislyn
miokardiale funksie het nie. Kreatienaanvulling het egter die miokardium se weerstand
teen iskemiese skade verlaag in harte van rotte blootgestel aan oefening: iskemiese
kontraktuur is verhoog en aorta-uitset tydens herperfusie is verlaag. Die presiese
meganismes hierby betrokke is egter onbekend en vereis dus verdere studie.Division of Medical Physiology (University of Stellenbosch), The National Research
Foundation and the Harry Crossley Fund for financial support