Am J Physiol Heart Circ Physiol 288: H3011-H3015, 2005.
First published February 4, 2005; doi:10.1152/ajpheart.00611.2004
0363-6135/05 $8.00
REPORTS
Fasudil prevents KATP channel-induced improvement in postischemic functional recovery
Kenya Nishizawa,2
Paul E. Wolkowicz,1
Tadashi Yamagishi,2
Ling-Ling Guo,1 and
Martin M. Pike1
1Department of Medicine, Division of Cardiovascular Disease, The University of Alabama at Birmingham, Birmingham, Alabama; and 2The National Defense Medical College, First Department of Medicine, Saitama, Japan
Submitted 21 June 2004
; accepted in final form 27 January 2005
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ABSTRACT
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Whereas activation of ATP-dependent potassium (KATP) channels greatly improves postischemic myocardial recovery, the final effector mechanism for KATP channel-induced cardioprotection remains elusive. RhoA is a GTPase that regulates a variety of cellular processes known to be involved with KATP channel cardioprotection. Our goal was to determine whether the activity of a key rhoA effector, rho kinase (ROCK), is required for KATP channel-induced cardioprotection. Four groups of perfused rat hearts were subjected to 36 min of zero-flow ischemia and 44 min of reperfusion with continuous measurements of mechanical function and 31P NMR high-energy phosphate data: 1) untreated, 2) pinacidil (10 µM) to activate KATP channels, 3) fasudil (15 µM) to inhibit ROCK, and 4) both fasudil and pinacidil. Pinacidil significantly improved postischemic mechanical recovery [39 ± 16 vs. 108 ± 4 mmHg left ventricular diastolic pressure (LVDP), untreated and pinacidil, respectively]. Fasudil did not affect reperfusion LVDP (41 ± 13 mmHg) but completely blocked the marked improvement in mechanical recovery that occurred with pinacidil treatment (54 ± 15 mmHg). Substantial attenuation of the postischemic energetic recovery was also observed. These data support the hypothesis that ROCK activity plays a role in KATP channel-induced cardioprotection.
cardioprotection; fasudil; pinacidil; rho kinase
OPENING CARDIAC ATP-SENSITIVE K+ (KATP) channels reduces infarct size, improves postischemic functional recovery, and is an important, required element in ischemic preconditioning (10). Despite its importance, the mechanisms responsible for KATP channel-induced cardioprotection remain elusive (10, 15, 16).
RhoA is a 21-kDa GTPase that regulates a variety of cellular processes involved with KATP channel-induced cardioprotection. However, the requirement for rhoA or its effectors, including rho kinase (ROCK), in KATP channel-induced cardioprotection, is unknown. The rhoA-signaling cascade regulates actin cytoskeletal dynamics (13). Notably, disrupting the myocyte actin cytoskeleton abrogates both KATP channel- and ischemic preconditioning-linked cardioprotection (3, 4), whereas morphological analyses show that the myocyte cytoskeleton is required to maintain sarcolemmal integrity during ischemia (2, 22). A possible mechanism is suggested by a report that cytochalasin D induces cardiac sarcolemmal KATP (sarcKATP) channel rundown and that F-actin restores channel activity (8). These results indicate the sarcKATP channel requires myocyte nonsarcomeric actin polymerization for its function. RhoA also directly activates myocyte phospholipase D (PLD), which protects cultured myocytes from damage during simulated ischemia (12). A recent report also indicates that rhoA is involved in mitochondrial reactive oxygen species signaling (24), a process that may be a required component of mitochondrial KATP (mitoKATP) channel-induced cardioprotection (10).
Thus we investigated the hypothesis that the activity of ROCK, a key effector of rhoA, is involved in KATP channel-induced cardioprotection. As one test of this hypothesis, we measured the extent to which fasudil, a pharmacologically effective, cell-permeable ROCK inhibitor (1, 6, 14, 19, 25), affects KATP channel cardioprotection in perfused rat hearts.
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MATERIALS AND METHODS
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Isolated perfused heart preparations.
All animal procedures were approved by and conformed to University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines. Hearts were isolated from male Sprague-Dawley rats (300–550 g), Langendorff perfused, and paced as previously described (7). Function was monitored via a left ventricular balloon and a Gould TA240 chart recorder, and was digitally recorded with a Digi-Med Heart Performance Analyzer. Hearts were initially perfused at 80 mmHg, 32°C, and pH 7.4 with a modified Krebs-Henseleit solution containing (in mM) 122 NaCl, 4.7 KCl, 1.2 MgSO4, 1.5 CaCl2, 25 NaHCO3, 0.5 Na2EDTA, and 11 glucose equilibrated with 95%O2-5% CO2. Flow was then set to that measured during a 15-min equilibration period, whereas left ventricular end-diastolic pressure was set to 10 mmHg.
Perfusion protocols.
All hearts were subjected to 36 min of zero-flow ischemia followed by 44 min of reperfusion at preischemic flow rates under four experimental protocols: untreated (n = 9), 10 µM pinacidil (n = 9), 15 µM fasudil (n = 7), and 15 µM fasudil + 10 µM pinacidil (n = 8). This concentration of fasudil was chosen based on the work of Davies et al. (6) who showed that 20 µM fasudil, also known as HA-1077, produced a 90% decrease in ROCK activity measured in vitro and that all other kinases tested were three to eight times less sensitive to fasudil than was ROCK. Additional reports from Altmann et al. (1), Sward et al. (19), and our laboratory (25) show that this concentration of fasudil effectively and selectively inhibits ROCK-linked processes in intact cardiac and smooth muscle preparations. Pinacidil (Sigma, St. Louis MO) was administered (0.01% final DMSO) for 16 min before and 2 min following ischemia, whereas aqueous fasudil (LC Labs, Woburn MA) was administered for 30 min before and 2 min following ischemia. Both agents were infused into the aortic line at a rate of 1% of total coronary flow. After all experiments, hearts were dried to a constant weight at 80°C and weighed.
NMR spectroscopy.
31P NMR spectra were continuously obtained (145.81 MHz, 60-degree excitation, 1.94-s recycle time, 2-min spectral acquisition) and spectral areas were quantified using NUTS software (Acorn NMR) (7). Saturation factors were as previously determined (7). Intracellular pH (pHi) was determined from the Pi frequency position (7). Spectral and functional data were averaged into 4-min time blocks and expressed as means ± SE.
Statistical analysis.
Differences across experimental groups were tested with general linear model ANOVA using Fisher's protected least significance difference method. Multiple comparisons across time were minimized by restricting the comparison to the time points noted in the figures.
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RESULTS
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Similar functional parameters (Table 1, Fig. 1A) were measured for all experimental groups during the initial normoxic perfusion period. Before ischemia, LVDP decreased slightly in fasudil-treated hearts but the difference from control hearts was not significant by the onset of ischemia (Fig. 1A, compare open squares vs. open circles). A series of normoxic hearts treated with fasudil showed this decrease to be completely reversible because their function returned to control levels within 4 min of fasudil washout (Fig. 1B). All experimental groups underwent similar degrees of ischemic contracture, and both pinacidil and fasudil + pinacidil-treated hearts had slightly delayed time to contracture (Table 1). At end reperfusion, the LVDP of untreated hearts was substantially decreased and the end-diastolic pressure was substantially increased compared with values measured before ischemia. Fasudil alone neither improved nor depressed reperfusion functional recovery. Consistent with its documented cardioprotective effects (7, 20), pinacidil increased both end-reperfusion LVDP and rate-pressure product to preischemic levels while substantially decreasing end-reperfusion end-diastolic pressure. Notably, however, all end-reperfusion functional parameters in hearts treated with fasudil + pinacidil were similar to those measured in control hearts and were significantly different from those measured in hearts treated with pinacidil alone (Fig. 1A).
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Table 1. Effect of fasudil, pinacidil, and fasudil + pinacidil on function of preischemic, ischemic, and reperfused rat hearts
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Fig. 1. A: Effect of fasudil, pinacidil, and fasudil + pinacidil on the mechanical function of ischemic-reperfused rat hearts. Left ventricular developed pressure (LVDP) means ± SE are reported across time for control (C,  ), fasudil-treated (F,  ), fasudil + pinacidil-treated (F + P,  ), and pinacidil treated (P,  ) hearts. Numbers of hearts and treatments are noted in MATERIALS AND METHODS. *P < 0.05 P vs. C;  P < 0.05 P vs. F;  P < 0.05 P vs. F + P; #P < 0.05 C vs. F, determined with general linear model (GLM) ANOVA using Fisher's protected least significant difference (LSD) test. B: effect of fasudil on LVDP in control perfused rat hearts. LVDP means ± SE are reported across time for a group of 3 hearts perfused with Krebs-Henseliet Glucose (KHG) and then with KHG containing 15 µM fasudil. Fasudil was removed from the perfusate at 0 min and function was monitored for an additional 80 min.
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Phosphocreatine (PCr) and ATP were similar in all four groups before ischemia (Fig. 2, A and B). After 23 min of ischemia, ATP was significantly lower in untreated hearts compared with the other three groups. At end ischemia, only pinacidil-treated hearts had slightly higher ATP levels compared with untreated hearts. Pi accumulation at end ischemia was significantly lower in pinacidil-treated compared with untreated hearts (45.0 ± 2.0 vs. 53.1 ± 2.4 µmol/g dry wt, respectively), whereas Pi in fasudil-treated hearts was similar to control (51.3 ± 3.4 µmol/g dry wt). End-ischemia Pi in fasudil + pinacidil-treated hearts (46.9 ± 2.2 µmol/g dry wt), although similar to that of pinacidil hearts, was not significantly different from untreated hearts.
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Fig. 2. A: effect of fasudil, pinacidil, and fasudil + pinacidil on the phosphocreatine content of ischemic-reperfused rat hearts. Phosphocreatine (PCr) means ± SE are reported across time for control, fasudil-treated, fasudil + pinacidil-treated, and pinacidil-treated hearts. Numbers of hearts and treatments are noted in MATERIALS AND METHODS. *P < 0.05 P vs. C; P < 0.05 P vs. F, determined with GLM ANOVA using Fisher's protected LSD test. B: effect of fasudil, pinacidil, and fasudil + pinacidil on the ATP content of ischemic-reperfused rat hearts. ATP means ± SE are reported across time for control, fasudil-treated, fasudil + pinacidil-treated, and pinacidil-treated hearts. Numbers of hearts and treatments are noted in MATERIALS AND METHODS. *P < 0.05 P vs. C; P < 0.05 P vs. F;  P < 0.05 C vs. F + P; #P < 0.05 C vs. F, determined with GLM ANOVA using Fisher's protected LSD. C: effect of fasudil, pinacidil, and fasudil + pinacidil on the intracellular pH of ischemic-reperfused rat hearts. LVDP means ± SE are reported across time for control, fasudil-treated, fasudil + pinacidil-treated, and pinacidil-treated hearts. Numbers of hearts and treatments are noted in MATERIALS AND METHODS. *P < 0.05 P vs. C; P < 0.05 P vs. F; P < 0.05 C vs. F + P, determined with GLM ANOVA using Fisher's protected LSD.
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The PCr and ATP levels measured in untreated hearts during reperfusion were lower than those measured before ischemia and similar to those measured in fasudil-treated hearts during reperfusion (Fig. 2, A and B). Consistent with its cardioprotective effect, pinacidil treatment markedly improved the reperfusion recovery of PCr and ATP compared with untreated hearts. Notably, reperfusion recovery of PCr and ATP in fasudil + pinacidil hearts was not different either from hearts treated with pinacidil alone or from untreated hearts, indicating that fasudil substantially attenuated the improvement in the energetic recovery induced by pinacidil. This was also reflected in the free energy of ATP hydrolysis, (
GATP, kJ/mol) calculated as previously described (20). The GATP at end reperfusion for untreated and fasudil-treated hearts was –53.1 ± 0.9 and –54.0 ± 0.6 kJ/mol, respectively, both significantly different from the value calculated for pinacidil hearts (–55.9 ± 0.2 kJ/mol). Analogous to PCr, although the GATP calculated for pinacidil + fasudil hearts (–54.7 ± 0.5 kJ/mol) was not significantly different from pinacidil-treated hearts, it clearly indicated a lower energy recovery than that of pinacidil hearts and was not significantly different from the GATP calculated for untreated hearts.
Measurements of pHi detected a slight lag in the development of ischemic acidosis in pinacidil-treated compared with untreated hearts, whereas fasudil did not alter pHi compared with untreated hearts at any time during ischemia (Fig. 2C). pHi was similar in all hearts at end ischemia. Only minor differences in pHi occurred between groups during reperfusion, with pinacidil-treated hearts having slightly higher reperfusion pHi than controls.
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DISCUSSION
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The cardioprotective effects of KATP channel opening are well documented but the effector mechanisms of cardioprotection are not fully elucidated (10, 15, 16). Here we demonstrate that a pharmacologically effective concentration of fasudil (6), a ROCK inhibitor (1, 6, 14, 19, 25), completely blocked the ability of pinacidil, a KATP channel opener, to enhance mechanical recovery during reperfusion. Consistent with this result, fasudil also substantially inhibited energetic recovery in pinacidil-treated hearts. Fasudil alone did not negatively affect either heart function or energetics during ischemia or reperfusion compared with untreated hearts. These data suggest that KATP cardioprotection requires ROCK activity but that inhibiting ROCK itself does not affect the ischemic damage observed in the absence of a KATP channel opener.
Data also suggest that fasudil inhibits energy recovery less completely than the mechanical recovery of postischemic pinacidil-treated hearts. Although the data do not conclusively dissociate functional and energetic protection, they suggest the possibility of separate cardioprotective components that may be differentially affected by ROCK inhibition. The existence of separate protective components rising from the different channel subtypes has been reported with the induction of cardioprotection by adenosine (21). In that model, the opening of the sarcKATP channel associated with the recovery of mechanical function, whereas opening the mitoKATP channel reduced infarct size. Pinacidil is known to activate both channel subtypes (17). Previous work by one of us (20) demonstrates that 5-hydroxydecanoate, a putative inhibitor of the mitoKATP channel, completely abolishes the beneficial effect of pinacidil on postischemic energy homeostasis and mechanical function as well as the ability of pinacidil to decrease lactate dehydrogenase release during reperfusion. Hence, ROCK modulation of effects originating from mitoKATP channel activation also may play a role in the current observations. The current study does not address the effect of ROCK inhibition on pinacidil-induced reductions of infarct size, a subject requiring further in-depth studies.
The subcellular location of the mitoKATP channel may lend itself to differentiation of the various components of KATP channel cardioprotection. Whereas mitoKATP channel activation may protect energy production via direct effects on mitochondrial volume (9) or mitochondrial Ca2+ uptake (15, 16, 23), other cardioprotective elements may depend on pathways downstream of mitoKATP. The action of rhoA/ROCK on these more distal pathways might offer an explanation for how differential effects of KATP channel-induced cardioprotection on mechanical function and energy homeostasis could occur. In support of this possibility, various studies (10, 17) report that cardioprotection following mitoKATP activation requires release of mitochondrial reactive oxygen species and a subsequent signal cascade involving kinases such as PLD and PKC. Involvement of rhoA/ROCK in such processes would be consistent with a recent report demonstrating the involvement of rhoA in mitochondrial reactive oxygen species signaling (24). It would also be consistent with the reported activation of PLD by rhoA in a myocyte model of adenosine-mediated cardioprotection. (12).
Regulation of actin cytoskeletal dynamics, an important, well-known function of rhoA/ROCK, is another mechanism that might link rhoA/ROCK signaling and KATP channel-induced cardioprotection. Baines et al. (3) and Cohen et al. (4) demonstrated that treatment with cytochalasin D, a specific disrupter of actin polymerization, abolished both ischemic preconditioning and mitoKATP channel-induced cardioprotection in myocytes. The mechanism by which this occurs has not been established. Interestingly, activation of the sarcKATP channel requires an intact cytoskeleton (8), suggesting the possibility that the cardioprotective role of the cytoskeleton may involve channel activation itself.
Work presented in this report provides the initial evidence that ROCK signaling may play a role in KATP channel-linked cardioprotection. The observation that fasudil does not affect the recovery of cardiac function during reperfusion in the absence of pinacidil, suggests three possible schemes of interaction between rhoA/ROCK and pinacidil-induced cardioprotection. Pinacidil may activate rhoA/ROCK signaling or protect the rhoA/ROCK signaling pathway from oxidative (5, 18) or proteolytic (11) damage during ischemia-reperfusion as part of the cardioprotective process. Alternatively, KATP channel activation itself may depend on rhoA/ROCK signaling (8). Distinguishing among these possibilities shall be the subject of future studies.
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GRANTS
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Support for this study was from an American Diabetes Association grant (to P. E. Wolkowicz), National Institutes of Health Grants HL-57965 (to P. E. Wolkowicz) and AA-12645 (to M. M. Pike), and from the National Defense Medical College, Japan (to K. Nishizawa and T. Yamagishi).
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ACKNOWLEDGMENTS
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We thank Dr. Ferdinand Urthaler for discussions suggesting this line of work and for careful reading of this manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. E. Wolkowicz, Dept. of Medicine, Univ. of Alabama at Birmingham, Birmingham, AL 35294 (E-mail:wolk{at}uab.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2005 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
