Ischaemic Preconditioning prevents the differentiation induced by ischaemia/reperfusion injury of rat cardiac fibroblast to myofibroblast
Kartika Pertiwi1and Lisa Chilton2
1: Biology Education Department, Faculty of Mathematics and Natural Science, Yogyakarta State University, Kampus UNY Karang Malang, Condong Catur, Depok, Sleman, Yogyakarta, 55281, Indonesia
2: Pharmacology and Physiology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland, 4811, Australia.
AIM
Ischaemia/reperfusion (IR) injury is a key source of myocardial damage in humans following acute myocardial infarction, post- cardiac arrest state and heart
transplantation (1, 2). The beneficial effects of ischaemic preconditioning (IPC) in minimising IR injury to cardiac muscle has been extensively studied, and involves activation of ATP-sensitive potassium (KATP) channels (3, 4). No previous studies have investigated the effects of IR injury or IPC in cardiac fibroblasts. Fibroblasts are responsible for maintaining the extracellular matrix in healthy hearts. Upon injury, they differentiate into the wound-healing phenotype, myofibroblasts, which may involve in maladaptive fibrosis. Our goals were to determine if: (a) IR injury evokes cardiac fibroblasts to differentiate into myofibroblasts; and if so, (b) does IPC ameliorate the IR-induced myofibroblast differentiation?
METHODS
Hearts were removed from isoflurane-anaesthetised adult rats, and fibroblasts dissociated by standard enzymatic digestion (5). Freshly dissociated fibroblasts were exposed to 30, 60 or 120 minutes of ischaemia by coating pelleted cells with sterile paraffin oil (modified from 6, 7), followed by 60 minutes of reperfusion when the oil was replaced with culture media. Fibroblasts were then placed under culture conditions and allowed to grow to ~70% confluency. Cultures were stained for expression of-smooth muscle actin, a marker of myofibroblast differentiation (8)
(immunohistology methods modified from 9, 10). RESULTS
IR of all durations evoked significant differentiation of fibroblasts into myofibroblasts, with 88±1% (mean±standard error, n=5) of cells differentiating following 30 minutes of ischaemia, 93±2% (n=4) following 60 minutes of ischaemia, and 92±1%(n=5) following 120 minutes of ischaemia. In contrast, time controls indicated that only 37±2% (n=5, 30 min.), 30±1% (n=5, 60 min.), and 45±2% (n=4, 120 min.) of cells differentiated from fibroblasts into myofibroblasts without IR. IPC produced by 15 minutes ischaemia and 30 minutes reperfusion, prior to 60 minutes of ischaemia and 60 minutes of reperfusion, significantly reduced the differentiation of fibroblasts into myofibroblasts from 88±2% (n=7, no IPC) to 46±4% (n=7, IPC)(Figure 1). The beneficial effect of IPC was blunted if cells were treated with 10 μ M glibenclamide, a KATPchannel blocker; in this protocol, 77±4% (n=8) of fibroblasts differentiated into myofibroblasts despite preconditioning (Figure 1).
CONCLUSION
These data indicate that IR injury strongly stimulates differentiation of cardiac fibroblasts into the wound-healing phenotype, the myofibroblast. IPC prevented IR-induced differentiation in a glibenclamide-sensitive manner, suggesting that
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Figure 1: Effect of ischaemia reperfusion injury, ischaemic preconditioning, and glibenclamide on rat cardiac fibroblast to myofibroblast differentiation. Following 60 minutes of ischaemia and 60 minutes of reperfusion, a significant percentage of fibroblasts were stimulated to differentiation into myofibroblasts, compared to time control. If cells were preconditioned with 15 minutes ischaemia and 30 minutes reperfusion prior to the 60 minutes ischaemia/60 minutes reperfusion, significantly fewer cells differentiated. Glibenclamide (10 μ M) significantly limited the beneficial effect of IPC.
Figure 2. This image presents a series of myofibroblasts expressing brown staining α-SMA, which polymerises to form stress fibres. The numbers illustrate the stress fibre grading system used in this study, which was: 0 (absent), 1 (occupying less than 25% of cell volume), 2 (occupying 26-50%), 3 (occupying 51-75%, not crossing the nucleus) and 4 (occupying >76% and/or crossing the nucleus). Cells not containing stress fibres were designated as fibroblasts, whereas cells containing stress fibres were designated as myofibroblasts. Myofibroblast differentiation ranged from proto-myofibroblasts (+1 to +3) and fully differentiated proto-myofibroblasts (Gabbiani et al., 1971; Skalli et al., 1986). Scale bar 50 µm, magnification: 400x.
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[image:3.611.163.450.391.600.2]Figure 3: Ischaemia and reperfusion is associated with fibroblast to myofibroblast differentiation. Exposing
freshly dissociated cardiac fibroblasts to ischaemia (30, 60 and 120 minutes) followed by 60 minutes reperfusion induced significantly more stress fibres to be expressed once cells were put into culture. Compared to time controls, 30 minutes (panel A), 60 minutes (panel B) and 120 minutes (panel C) ischaemia reduced the number of fibroblasts in culture (staining intensity 0), while evoking differentiation into proto-myofibroblasts (staining intensities 1-3) and fully differentiated myofibroblasts (staining intensity 4). *:P<0.05, compared to time control.
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