Ischaemic Preconditioning prevents the differentiation induced byischaemia/reperfusion injury of rat cardiac fibroblast to myofibroblast

Kartika Pertiwi1 and 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, Indonesia2: Pharmacology and Physiology, School of Veterinary and Biomedical Sciences,James Cook University, Townsville, Queensland, 4811, Australia.

AIMIschaemia/reperfusion (IR) injury is a key source of myocardial damage in humansfollowing acute myocardial infarction, post- cardiac arrest state and hearttransplantation (1, 2). The beneficial effects of ischaemic preconditioning (IPC) inminimising IR injury to cardiac muscle has been extensively studied, and involvesactivation of ATP-sensitive potassium (KATP) channels (3, 4). No previous studieshave investigated the effects of IR injury or IPC in cardiac fibroblasts. Fibroblasts areresponsible for maintaining the extracellular matrix in healthy hearts. Upon injury,they differentiate into the wound-healing phenotype, myofibroblasts, which mayinvolve in maladaptive fibrosis. Our goals were to determine if: (a) IR injury evokescardiac fibroblasts to differentiate into myofibroblasts; and if so, (b) does IPCameliorate the IR-induced myofibroblast differentiation?METHODSHearts were removed from isoflurane-anaesthetised adult rats, and fibroblastsdissociated by standard enzymatic digestion (5). Freshly dissociated fibroblasts wereexposed to 30, 60 or 120 minutes of ischaemia by coating pelleted cells with sterileparaffin oil (modified from 6, 7), followed by 60 minutes of reperfusion when the oilwas replaced with culture media. Fibroblasts were then placed under cultureconditions and allowed to grow to ~70% confluency. Cultures were stained forexpression of -smooth muscle actin, a marker of myofibroblast differentiation (8)(immunohistology methods modified from 9, 10).RESULTSIR of all durations evoked significant differentiation of fibroblasts into myofibroblasts,with 88±1% (mean±standard error, n=5) of cells differentiating following 30 minutesof 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 only37±2% (n=5, 30 min.), 30±1% (n=5, 60 min.), and 45±2% (n=4, 120 min.) of cellsdifferentiated from fibroblasts into myofibroblasts without IR. IPC produced by 15minutes ischaemia and 30 minutes reperfusion, prior to 60 minutes of ischaemia and60 minutes of reperfusion, significantly reduced the differentiation of fibroblasts intomyofibroblasts from 88±2% (n=7, no IPC) to 46±4% (n=7, IPC)(Figure 1). Thebeneficial effect of IPC was blunted if cells were treated with 10 μM glibenclamide, aKATP channel blocker; in this protocol, 77±4% (n=8) of fibroblasts differentiated intomyofibroblasts despite preconditioning (Figure 1).CONCLUSIONThese data indicate that IR injury strongly stimulates differentiation of cardiacfibroblasts into the wound-healing phenotype, the myofibroblast. IPC prevented IR-induced differentiation in a glibenclamide-sensitive manner, suggesting thatactivation of KATP channels is part of the mechanism by which IPC protects cardiacfibroblasts. (=2831 characters + spaces; 3000 = limit)

REFERENCES1. Park J L and Lucchesi B R (1999) Mechanisms of myocardial reperfusion injury.

Annual Thoracic Surgery 68:1905–19122. Piper H M, Meuter K and Schaefer C (2003) Cellular mechanisms of ischemia-

reperfusion injury. Annual Thoracic Surgery 75: 644-6483. Murray et al. (1986) Preconditioning with ischaemia: a delay of lethal cell injury in

ischaemic myocardium. Circulation 74: 1124-11364. Gross G J and Auchampach J A (1992) Blockade of ATP-sensitive potassium

channels prevents myocardial preconditioning in dogs. Circulation Research70: 223–233

5. Chilton L, Ohya S, Freed D, George E, Drobic V, Shibukawa Y, MacCannell Y,Imaizumi R B, Clark I, Dixon M and Giles W R (2005) K+ currents regulate theresting membrane potential, proliferation, and contractile responses inventricular fibroblasts and myofibroblasts. American Journal of PhysiologyHeart Circulation Physiology 288: H2931–H2939.

6. Lai Z F and Nishi K (1998) Intracellular chloride activity increases in guinea pigventricular muscle during simulated ischemia. American Journal of Physiology(Heart Circulation) 275(44): H1613–H1619

7. Han J, Kim E, Ho W K and Earm YE (1996) Blockade of the ATP-sensitivepotassium channel by taurine in rabbit ventricular myocytes. Journal ofMolecular Cell Cardiology 28(9):2043-2050

8. Tomasek J J, Gabbiani G, Hinz B, Chponnier C and Brown R A (2002)Myofibroblasts and mechano-regulation of connective tissue remodeling.Molecular Cell Biology 3: 349-363

9. Raizman J E, Komljenovic J, Chang R, Deng C, Bedosky K M, Rattan S G,Cunnington R H, Freed D H and Dixon I M C (2007) The participation of theNa2+ - Ca2+ exchanger in primary cardiac myofibroblast migration, contractionand proliferation. Journal of Cellular Physiology 213: 540-551

10. Gorton et al.( 2009) B- and T-Cell responses in group of a streptococcus m-protein- or peptide-induced experimental carditis Infection and Immunity 77(5):2177-2183

Figure 1: Effect of ischaemia reperfusion injury, ischaemic preconditioning, andglibenclamide on rat cardiac fibroblast to myofibroblast differentiation. Following 60minutes of ischaemia and 60 minutes of reperfusion, a significant percentage offibroblasts were stimulated to differentiation into myofibroblasts, compared to timecontrol. If cells were preconditioned with 15 minutes ischaemia and 30 minutesreperfusion prior to the 60 minutes ischaemia/60 minutes reperfusion, significantlyfewer cells differentiated. Glibenclamide (10 μM) significantly limited the beneficialeffect of IPC.

Figure 2. This image presents a series of myofibroblasts expressing brown stainingα-SMA, which polymerises to form stress fibres. The numbers illustrate the stressfibre grading system used in this study, which was: 0 (absent), 1 (occupying less than25% of cell volume), 2 (occupying 26-50%), 3 (occupying 51-75%, not crossing thenucleus) and 4 (occupying >76% and/or crossing the nucleus). Cells not containingstress fibres were designated as fibroblasts, whereas cells containing stress fibreswere designated as myofibroblasts. Myofibroblast differentiation ranged from proto-myofibroblasts (+1 to +3) and fully differentiated myofibroblasts (Gabbiani et al.,1971; Skalli et al., 1986). Scale bar 50 µm, magnification: 400x.

0

3

2

1

4 0

3

2

1

4

Figure 3: Ischaemia and reperfusion is associated with fibroblast to myofibroblastdifferentiation. Exposingfreshly dissociated cardiac fibroblasts to ischaemia (30, 60 and 120 minutes)followed by 60 minutes reperfusion induced significantly more stress fibres to beexpressed 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 thenumber of fibroblasts in culture (staining intensity 0), while evoking differentiation intoproto-myofibroblasts (staining intensities 1-3) and fully differentiated myofibroblasts(staining intensity 4). *: P<0.05, compared to time control.

A

B

C

Figure 4: The degree of differentiation is sensitive to the length of the ischaemicinsult.