Cardio-protective role of ghrelin and synthetic analogues in the in vitro ischemia/reperfusion mouse heart model and the underlying molecular mechanism

Ma, Yi (2012). Cardio-protective role of ghrelin and synthetic analogues in the in vitro ischemia/reperfusion mouse heart model and the underlying molecular mechanism PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Ma, Yi
Thesis Title Cardio-protective role of ghrelin and synthetic analogues in the in vitro ischemia/reperfusion mouse heart model and the underlying molecular mechanism
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Chen Chen
Bradley S. Launikonis
Total pages 140
Total colour pages 30
Total black and white pages 110
Language eng
Subjects 110201 Cardiology (incl. Cardiovascular Diseases)
110299 Cardiovascular Medicine and Haematology not elsewhere classified
111699 Medical Physiology not elsewhere classified
Formatted abstract
Ischemic heart disease is a leading cause of mortality in the world and reperfusion is the current major therapy for it. Ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage during ischemia and subsequent recovery of cardiac coronary blood supply during reperfusion and to study the I/R injury observed in the patients after cardiac surgery and potential therapy for it. Pharmacological intervention has been developed to reduce the I/R injury but with limited protective effects. Side effects and incomplete mechanisms prevent the clinical application of these drugs, including growth hormone secretagogues (GHS). Previous work showed inconsistent results of GHS (ghrelin and its synthetic analogue hexarelin) on the I/R cardiac models, and the effective pathways of them are still not quite clear. In particular, the role of GHS in the regulation of intracellular Ca2+ concentration ([Ca2+]i) imbalance and oxidative stress essential for the I/R injury is not totally understood. The aim of this project is to determine whether the presence of GHS would protect cardiomyocyte function from I/R injury and to examine the underlying mechanisms including regulation of [Ca2+]i and other ion currents, and the oxidative stress related mitogen-activated protein kinase (MAPK) pathway.

In order to address this issue, global ischemia was induced in isolated Langendorff hearts from adult male C57BL mice by stopping the perfusion through the aorta for 20 min, followed by 30 min of perfusion, whereas control group hearts were continuously perfused. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system right before or after ischemia for 10 min, termed pre- or post-treatment. Cardiomyocytes were freshly isolated from these hearts and parameters including cell shortening, [Ca2+]i transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ were measured. Whole-cell patch-clamp recording technique was used to measure action potential (AP), voltage-gated L-type calcium current (ICaL), transient outward potassium current (Ito) and sodium current (INa). In addition, conventional RT-PCR was used to examine the mRNA expression level of GHS receptor type 1a (GHS-R1a) in the heart. Western blots were used to estimate the expression ratio of phosphorylated protein to the total protein, including phosphorylated phospholamban (p-PLB)/total phospholamban (PLB), which is essential for the SR Ca2+ ATPase (SERCA) activity, and important kinases in the MAPK pathway, for example ratios of phosphorylated to total extracellular signal-regulated kinase (ERK) involved in stimulating proliferation and cellular surviving, p38 and c-jun N-terminal kinases (JNK) involved in triggering apoptosis.

Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening and [Ca2+]i transient amplitude after I/R through regulation of [Ca2+]i by recovery of ICaL and SR Ca2+ content via maintaining a normal ratio of p-PLB/PLB and therefore SERCA activity. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca2+]i transients. The AP amplitude, maximal upstroke velocity (Vmax), and AP duration (APD) at 90% repolarization were significantly decreased by I/R, which were maintained at control levels by GHS pre- or post-treatments. The decreased amplitude of AP after ischemia was partially due to the reduced INa. Ghrelin and hexarelin pre- and post-treatments prevented the decrease in INa from I/R injury. The significant increase in Ito after I/R partially explained the shortened APD, and GHS treatments restored APD by reducing the Ito after I/R to the control level. In addition, GHS treatments could also shift the half maximum inactivation voltage of Ito to more negative voltages leading to a decreased availability of Ito. Western blots showed that the significant increased ratios of phosphorylated to total protein of phosphorylated ERK (p-ERK), phosphorylated p38 (p-p38) and phosphorylated JNK (p-JNK) in the MAPK pathway following I/R, reflecting the increased cell death and cell surviving. GHS treatments inhibited the exaggerated activation of JNK and p38, but further increased the phosphorylation of ERK, indicating the inhibition of apoptosis and activation of cellular surviving pathways, all of which were reduced or eliminated by the BIM28163, suggesting the activation of GHS-R1a in these effects of GHS on MAPK pathway.

In conclusion, through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes with protecting them from I/R injury, probably through protecting or recovering ICaL and the ratio of p-PLB/PLB and then SR Ca2+ content to allow the maintenance or recovery of normal cardiac contractility. In addition, GHS also preserved the AP properties of cardiomyocytes from I/R injury via modification of trans-membrane voltage-gated ionic currents (Ito and INa), inhibition of apoptosis (p-p38/p38 and p-JNK/JNK) and activation of cellular surviving pathways (p-ERK/ERK), which were at least partially mediated by GHS-R1a. These observations provide supporting evidence to the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury.
Keyword Ischemia/reperfusion
Growth hormone secretagogue
Action potential
Voltage-gated current
Cardioprotective effect
Growth hormone secretagogue receptor

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Created: Tue, 09 Oct 2012, 20:33:02 EST by Yi Ma on behalf of Scholarly Communication and Digitisation Service