Ischaemia Preconditioning Prevents IR-induced Myofibroblast Differentiation
Ischaemic Preconditioning prevents the differentiation induced by
ischaemia/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, 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
KATP channel 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 IRinduced differentiation in a glibenclamide-sensitive manner, suggesting that
activation of KATP channels is part of the mechanism by which IPC protects cardiac
fibroblasts. (=2831 characters + spaces; 3000 = limit)
REFERENCES
1. Park J L and Lucchesi B R (1999) Mechanisms of myocardial reperfusion injury.
Annual Thoracic Surgery 68:1905–1912
2. Piper H M, Meuter K and Schaefer C (2003) Cellular mechanisms of ischemiareperfusion injury. Annual Thoracic Surgery 75: 644-648
3. Murray et al. (1986) Preconditioning with ischaemia: a delay of lethal cell injury in
ischaemic myocardium. Circulation 74: 1124-1136
4. Gross G J and Auchampach J A (1992) Blockade of ATP-sensitive potassium
channels prevents myocardial preconditioning in dogs. Circulation Research
70: 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 the
resting membrane potential, proliferation, and contractile responses in
ventricular fibroblasts and myofibroblasts. American Journal of Physiology
Heart Circulation Physiology 288: H2931–H2939.
6. Lai Z F and Nishi K (1998) Intracellular chloride activity increases in guinea pig
ventricular 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-sensitive
potassium channel by taurine in rabbit ventricular myocytes. Journal of
Molecular 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 the
Na2+ - Ca2+ exchanger in primary cardiac myofibroblast migration, contraction
and proliferation. Journal of Cellular Physiology 213: 540-551
10. Gorton et al.( 2009) B- and T-Cell responses in group of a streptococcus mprotein- or peptide-induced experimental carditis Infection and Immunity 77(5):
2177-2183
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.
3
2
1
4
0
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 protomyofibroblasts (+1 to +3) and fully differentiated myofibroblasts (Gabbiani et al.,
1971; Skalli et al., 1986). Scale bar 50 µm, magnification: 400x.
A
B
C
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
ischaemia/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, 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
KATP channel 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 IRinduced differentiation in a glibenclamide-sensitive manner, suggesting that
activation of KATP channels is part of the mechanism by which IPC protects cardiac
fibroblasts. (=2831 characters + spaces; 3000 = limit)
REFERENCES
1. Park J L and Lucchesi B R (1999) Mechanisms of myocardial reperfusion injury.
Annual Thoracic Surgery 68:1905–1912
2. Piper H M, Meuter K and Schaefer C (2003) Cellular mechanisms of ischemiareperfusion injury. Annual Thoracic Surgery 75: 644-648
3. Murray et al. (1986) Preconditioning with ischaemia: a delay of lethal cell injury in
ischaemic myocardium. Circulation 74: 1124-1136
4. Gross G J and Auchampach J A (1992) Blockade of ATP-sensitive potassium
channels prevents myocardial preconditioning in dogs. Circulation Research
70: 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 the
resting membrane potential, proliferation, and contractile responses in
ventricular fibroblasts and myofibroblasts. American Journal of Physiology
Heart Circulation Physiology 288: H2931–H2939.
6. Lai Z F and Nishi K (1998) Intracellular chloride activity increases in guinea pig
ventricular 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-sensitive
potassium channel by taurine in rabbit ventricular myocytes. Journal of
Molecular 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 the
Na2+ - Ca2+ exchanger in primary cardiac myofibroblast migration, contraction
and proliferation. Journal of Cellular Physiology 213: 540-551
10. Gorton et al.( 2009) B- and T-Cell responses in group of a streptococcus mprotein- or peptide-induced experimental carditis Infection and Immunity 77(5):
2177-2183
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.
3
2
1
4
0
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 protomyofibroblasts (+1 to +3) and fully differentiated myofibroblasts (Gabbiani et al.,
1971; Skalli et al., 1986). Scale bar 50 µm, magnification: 400x.
A
B
C
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