Ranolazine as a Cardioplegia Additive Improves Recovery of Diastolic Function in Isolated Rat Hearts
Background— Ranolazine (Ran), an antianginal agent, inhibits late Na+ current. The purpose of this study was to determine whether there was an added benefit of adding Ran to cardioplegia (CP) in a model of global ischemia/reperfusion.
Methods and Results— Isolated rat hearts were Langendorff-perfused and exposed to 40-minute normothermic, cardioplegic global ischemia and 30 minutes of reperfusion. Before ischemia and during reperfusion, hearts were treated with no drug (control) or with the late Na+ current inhibitors Ran (5 μmol/L) or tetrodotoxin (1 μmol/L). Ischemic cardioplegic arrest led to an increase of left ventricular end-diastolic pressure (LVEDP) by ≥20 mm Hg (ie, cardiac contracture). Ten out of 11 hearts treated with CP alone developed contracture, whereas 6 out of 11 hearts treated with CP plus Ran developed contracture. Ran added to CP reduced LVEDP at the end of ischemia from 41±5 mm Hg in CP alone to 26±3 mm Hg in CP plus Ran (P=0.024). Area under the curve for LVEDP during the entire ischemic period was also smaller in CP plus Ran versus CP alone. The percent increase (from baseline) of LVEDP measured at the end of 30-minute reperfusion was smaller for CP plus Ran (66±18%) versus CP alone (287±90%; P=0.035). The area under the curve for LVEDP during reperfusion was smaller in CP plus Ran versus CP alone. Tetrodotoxin (1 μmol/L) also reduced cardiac contracture during ischemia/reperfusion, compared to CP alone.
Conclusions— Our results suggest that Ran may have therapeutic potential as an adjunct to CP and further support a protective role of Na+ current inhibition during ischemia/reperfusion.
Although cardioplegia (CP) solution provides protection against ischemic cardiac arrest during surgery, enhancements in cardioplegic preservation methods are needed to match the increasing complexity of operations in the contemporary era. For most surgeons, a crystalloid solution admixed with blood forms the basic CP composition, to which pharmacological agents are added. Results of addition of Krebs cycle intermediates,1,2 L-arginine,3 and nicorandil3,4 to CP have resulted in mixed findings. Because inhibition of cardiac late Na+ current (late INa) has been shown to be cardioprotective,5,6 we determined if the addition of a late INa inhibitor improved cardioplegic preservation of contractile function. Abnormally slow inactivation of Na+ channel current caused by ischemia leads to increased late INa and results in accumulation of cellular Na+.7–9 Na+ overload leads to increased reverse mode Na+-Ca2+ exchange and Ca2+ overload.9 Ranolazine is an FDA-approved antianginal, anti-ischemic agent that inhibits late INa.10,11 By blocking late INa, ranolazine can reduce the amount of Na+ that can be exchanged for Ca2+ via the Na+-Ca2+ exchanger and, subsequently, attenuate intracellular Ca2+ overload,10,12 resulting in reduced left ventricular end-diastolic pressure (LVEDP) and improved ventricular relaxation.11,13 Compared to pharmacological manipulations of CP that produce hypotension, clinical experience with ranolazine as an antianginal agent has shown that it has little or no effect on blood pressure and heart rate.13,14 The purpose of this study was to investigate the potential use of ranolazine as an adjunct to CP and its protective role in the setting of potassium-based cardioplegic arrest. The effect of tetrodotoxin (TTX), a selective inhibitor of Na+ channel current, was also determined to confirm that inhibition of Na+ channel current was beneficial during cardioplegic arrest.
Materials and Methods
Isolated Langendorff-Perfused Heart
Hearts isolated from female Sprague-Dawley rats (≈2 months old; Charles River Laboratories, Hollister, Calif) were Langendorff-perfused. Rats were anesthetized with sodium pentobarbital (40 to 80 mg/kg, intraperitoneal) and heparinized (1000 U/kg, intraperitoneal). Hearts were quickly excised under full anesthesia and placed in prechilled (4°C) Krebs-Henseleit bicarbonate buffer (KHB), then cannulated through the aorta. The cannula was connected to a Langendorff apparatus and hearts were perfused with KHB solution (pH 7.4, 36°C, 95% O2 plus 5% CO2).
Measurements of Contractile Function
After equilibration, a distilled water-filled polyvinyl-chloride balloon was placed on the tip of a catheter and inserted into the left ventricular cavity. LVEDP was adjusted to ≈5 mm Hg by filling the fluid-filled balloon with distilled water. The balloon-tipped catheter was connected to a pressure transducer for measurement of intraventricular pressure. Data were collected using an AD Instruments (Grand Junction, Colo) acquisition system, and LVEDP and maximal rates of pressure increase (+dP/dtmax) and decrease (−dP/dtmax) were calculated. Coronary flow was measured by timed collection of the pulmonary artery effluent. The hearts were paced at a rate of ≈300 beats per minute. Indices of contractile function were measured before ischemia and at the end of the 30-minute reperfusion period. Cardiac contracture caused by ischemia/reperfusion (I/R) and the protective effects of CP and ranolazine were examined by measuring the time to development of 20 mm Hg of LVEDP during ischemia. The extent of reperfusion-induced contracture was determined by measuring LVEDP at the end of the 30-minute reperfusion period. Contracture was also assessed by quantification of the estimated area under the curve analysis for LVEDP during the entire ischemic and reperfusion period.
Experimental Protocols and Groups
Two sets of I/R protocols (Figure 1) were performed. In both protocols, coronary perfusion with KHB was performed for 30 minutes and then terminated to induce global ischemia. CP was given at the onset and at 20 minutes into the ischemic period. Ischemia was followed by a 30-minute reperfusion. The ischemic interval of 40 minutes was selected to approximate the clinical time needed for construction of several coronary bypass grafts and was sufficient to impair contraction. Hearts were submerged in buffer during experiments to maintain normothermia.
To investigate the potential use of ranolazine as an adjunct to CP and the specific role of inhibition of late INa, hearts subjected to I/R were assigned to 2 different groups: I/R CP treatment without (CP alone; n=11) and with 5 μmol/L ranolazine (n=11; protocol 1). The 5-μmol/L dose of ranolazine is a low dose that inhibits late INa but is unlikely to affect other ion channel currents.15 To confirm that reduction of Na+ influx is cardioprotective during I/R, 1 μmol/L TTX, a known blocker of Na+ influx, was used in a second series of experiments (protocol 2).
The composition of KHB solution was (in mmol/L): 118 NaCl, 10 glucose, 4.7 KCl, 2.5 CaCl2, 1.18 KH2PO4, 1.2 MgSO4, and 25 NaHCO3. The composition of CP solution (low-potassium Fremes) was: 0.45% normal saline with 18 mEq/L MgSO3, 100 mEq/L KCl, 12 mEq/L tromethamine, and 20 mL citrate phosphate dextrose solution (pH 7.95).
Data are presented as mean±SE. Differences in means between 2 experimental groups were analyzed using 2-sample t tests and confirmed using Kruskal-Wallis (which does not assume normally distributed data). If the Kruskal-Wallis test did not confirm the t test results, then the Kruskal-Wallis P value was reported (and noted with K-W). Equality of variances was tested using the Folded F-test (SAS Version 9.1; SAS, Cary, NC), and if the hypothesis of equal variances was rejected (P<0.1), then the t test using Satterthwaite approximation was used. The areas under the curves were calculated by ImageJ software (NIH, version 1.38). P<0.05 was considered significant. Differences in proportions were tested using Fisher’s exact test.
Statement of Responsibility
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Effects of 5 μmol/L Ranolazine on Cardiac Contracture During Ischemia
To investigate a therapeutic use of ranolazine as an adjunct to CP and a specific role of late INa inhibition in a model of cardioplegic I/R, a 5-μmol/L dose of ranolazine was used to pretreat hearts before ischemia, and as a supplement to CP solution. There was no significant difference in LVEDP in the absence and presence of ranolazine at baseline during the 15 minutes of perfusion of hearts with KHB solution (1.9±0.1 versus 2.4±0.5 mm Hg, respectively; P=0.330; Table 1). Ranolazine attenuated the I/R-induced increase of LVEDP. LVEDP measured at the end of ischemia was significantly lower in hearts treated with CP plus ranolazine (26±3 mm Hg; n=11) compared to hearts receiving CP alone (41±5 mm Hg; n=11; P=0.024). Six out of 11 hearts treated with CP plus ranolazine reached an LVEDP of 20 mm Hg during ischemia, whereas 10 out of 11 hearts treated with CP alone reached LVEDP of 20 mm Hg (not significantly different; P=0.149). The entire ischemic area under the curve for LVEDP was significantly smaller in hearts treated with CP plus ranolazine versus CP alone (P=0.019; Figure 2A).
Effects of 5 μmol/L Ranolazine on Recovery of Postischemic Contractile Function
The percent increase in LVEDP from baseline to the end of 30-minute reperfusion was significantly smaller in CP plus ranolazine (66±18%) versus CP alone (287±90%; P=0.035; Table 1). Area under the curve of the increase of LVEDP during the entire reperfusion phase was also significantly smaller in hearts receiving ranolazine than in hearts without ranolazine (P=0.036; Figure 2B). Values of +dP/dtmax measured at the end of 30-minute reperfusion were not statistically improved (P=0.112) by addition of ranolazine (Table 1). There was a nonsignificant trend (P=0.087) toward better recovery of −dP/dtmax in CP plus ranolazine hearts, compared to CP alone (Table 1). The percent decrease in coronary flow at 30 minutes of reperfusion was reduced (that is, improved) in CP plus ranolazine hearts compared to CP alone (P=0.017; Table 1).
Effects of 1 μmol/L TTX on Cardiac Contracture During Ischemia
There was no significant difference in LVEDP in the absence and presence of TTX at baseline during the 15 minutes of perfusion of hearts with KHB solution (2.3±0.5 versus 1.8±0.3 mm Hg, respectively; P=0.381; Table 2). All hearts treated with CP alone (10/10 hearts) and 7 of 9 hearts treated with TTX reached contracture (an LVEDP of 20 mm Hg) during ischemia (not significantly different; P=0.211). However, LVEDP measured at the end of ischemia was significantly lower in hearts in which CP was supplemented with TTX (27±2 mm Hg) versus CP alone (38±4 mm Hg; P=0.020). The entire ischemic area under the curve of the increase of LVEDP was also smaller in hearts treated with CP plus TTX than in hearts treated with CP alone (P=0.048; Figure 3A).
Effects of 1 μmol/L TTX on Recovery of Postischemic Contractile Function
The percent increase of LVEDP from baseline to the end of reperfusion was significantly lower in hearts treated with TTX plus CP than in hearts treated with CP alone (P=0.021; K-W; Table 2). The area under the curve of the increase of LVEDP during the entire reperfusion phase was significantly smaller in hearts receiving TTX than in hearts with CP alone (P=0.046; Figure 3B). Values of +dP/dtmax and −dP/dtmax measured at the end of 30-minute reperfusion (Table 2) were not significantly improved by the addition of TTX (P=0.339 and P=0.290, respectively, versus CP alone). There was a nonsignificant trend toward greater coronary flow at 30 minutes of reperfusion in CP plus TTX hearts compared to CP alone (Table 2).
We investigated the effect of ranolazine to improve cardiac contractile function in a protocol wherein rat isolated hearts were exposed to global ischemia in the presence of cardioplegia solution, followed by reperfusion. A therapeutic concentration of ranolazine (5 μmol/L), at which the drug is a relatively selective inhibitor of late INa,12 was used. The major findings of our study were that inclusion of 5 μmol/L ranolazine to cardioplegia solution resulted in less contracture during ischemia and a smaller elevation in LVEDP during reperfusion, compared to CP alone. Treatment of hearts with the selective INa inhibitor TTX (1 μmol/L) also significantly reduced the increases of LVEDP during ischemia and reperfusion, compared to CP alone. These results suggest that inhibition of the late INa may benefit the ischemic, cardioplegia-arrested heart by reducing development of ventricular contracture during ischemia and by reducing ventricular diastolic wall tension during reperfusion. In addition, there was a trend to increased coronary flow during reperfusion in ranolazine-treated hearts. We speculate that the lower diastolic tension in ranolazine-treated hearts may allow better perfusion of subendocardial myocardium, thus leading to the observed trend to increased coronary flow in these hearts. Because continuous perfusion of CP is used in cardiac surgical practice, a reduction of diastolic wall tension and an increase of coronary perfusion may lead to improved access of CP to myocardium and better cardioprotection.
Various studies of animal models of I/R have provided evidence that late INa is increased during I/R and that, coupled with enhanced Na+-Ca2+ exchanger activity, promotes cytosolic Ca2+ overload, induces contracture, elevates LVEDP, and impairs cardiac contractile function.9,16,17 Treatment with ranolazine, a selective inhibitor of the late INa, delays cardiac contracture and reduces the LVEDP elevation and diastolic Ca2+ overloading during I/R, suggesting an important role of enhanced late INa as a cause of ischemia-induced myocardial damage and the protective effect of the inhibition of the late INa during I/R.9,16
Using 50 μmol/L ranolazine, we previously showed that adding ranolazine to CP attenuates ischemic contracture and contracture occurring during reperfusion.18 The interpretation of that study was limited by the fact that ranolazine at a high concentration of 50 μmol/L is not a selective inhibitor of late INa. In this study, the effects of 5 μmol/L ranolazine (a concentration within the therapeutic range and that is associated with selective inhibition of late INa)15 and the effects of TTX, a specific inhibitor of Na+ channel current, were studied to better identify the mechanism of the protective effect of ranolazine. Ranolazine inhibits the late INa with an IC50 value of <6 μmol/L.15 At higher concentrations, ranolazine also inhibits other depolarizing and repolarizing currents, including IKr (IC50 12 μmol/L), peak INa (IC50 240 μmol/L), ICaL (IC50 296 μmol/L), late ICaL (IC50 50 μmol/L), and INa-Ca (IC50 91 μmol/L).15,19 Inhibition of the late INa and Na+ influx is thus the predominant effect of <6 μmol/L ranolazine, and the effect most likely to explain its cardiac protective action in the present study.
Various pharmacological interventions have been tested as adjunctive therapies to further improve cardioplegic protection and slow the process of myocardial ischemic injury, but the results of these studies have been inconsistent. Glucose-insulin CP solution has been used to augment adenosine 5′-triphosphate formation in energy-deprived hearts. Although promising results were initially reported,20 the existing evidence does not support the efficacy of glucose-insulin in patients at high risk requiring myocardial revascularization.21 The use of β-adrenergic blockade as an adjunct to CP was advocated to reduce myocardial oxygen consumption,22 but clinical application could be complicated by long-lasting, depressing effects of β-adrenergic blockade on cardiac contractility and heart rate. Hyperpolarizing agents such as the K+ channel opener nicorandil have been proposed as an alternative to depolarizing CP.23 They arrest the heart at a membrane potential close to or more negatively than the resting potential. This approach theoretically reduces perturbation of ionic balance and maintains cellular homeostasis. Although early clinical success had been reported, nicorandil was shown to be harmful, and even to cause hypotension and arrhythmias.4 In contrast, in clinical trials, ranolazine has minimal effects on heart rate and blood pressure.13,14
This study investigated cardioprotective effect of ranolazine using a small rodent model (Charles River Laboratories, Hollister, Calif). In rat cardiac myocytes, the level of cellular Na+ is much higher under normal conditions than that in rabbit, guinea pig, or human cardiac cells.24 Differences in cellular Na+ handling among species25 could potentially confound an estimation of the significance in the human of the enhanced late INa during I/R and its inhibition by ranolazine in the rat. Second, although the present study investigated the efficacy of ranolazine using a low concentration (5 μmol/L) that is known to specifically inhibit the late INa, we cannot entirely rule out the possibility that ranolazine had small inhibitory effects on other ionic currents and that these effects contributed to protection of the ischemic heart. Finally, in this study, the protective effect of ranolazine in a normothermic crystalloid CP solution was determined, and the results should not be extrapolated to the model of cold-blood CP (that is the most commonly used myocardial protective strategy in today’s cardiac surgery) without further investigation.
In conclusion, using a clinically relevant dose of ranolazine, the present study demonstrates a new therapeutic potential of ranolazine as an adjunct to cardioplegia, as evidenced by reduced myocardial contracture during ischemia, attenuation of the reperfusion-induced increase of LVEDP, and better preservation of coronary flow.
The authors thank their statistician, Lois Kelleman, who has helped with statistical analysis.
Sources of Funding
This study was supported by a research grant from Cardiovascular Therapeutics, Inc (currently, Gilead).
L. Belardinelli, A.K. Dhalla, and J.C. Shryock are currently employees of Gilead, Inc. Dr Kloner is a speaker and consultant for and has received grant support from Cardiovascular Therapeutics, Inc (currently, Gilead).
Presented in part at American Heart Association Scientific Sessions 2008, November 8–12, 2008, New Orleans, La.
Greenwood JP, Malik I, Jennings P, Stevenson RN. Haemodynamic and electrocardiographic consequences of severe nicorandil toxicity. Emerg Med J. 2003; 20: 98–100.
Ver Donck L, Borgers M, Verdonck F. Inhibition of sodium and calcium overload pathology in the myocardium: a new cytoprotective principle. Cardiovasc Res. 1993; 27: 349–357.
Murphy E, Cross H, Steenbergen C. Sodium regulation during ischemia versus reperfusion and its role in injury. Circ Res. 1999; 84: 1469–1470.
Imahashi K, Kusuoka H, Hashimoto K, Yoshioka J, Yamaguchi H, Nishimura T. Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury. Circ Res. 1999; 84: 1401–1406.
Song Y, Shryock JC, Wagner S, Maier LS, Belardinelli L. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J Pharmacol Exp Ther. 2006; 318: 214–222.
Haigney MC, Lakatta EG, Stern MD, Silverman HS. Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation. 1994; 90: 391–399.
Chaitman BR, Pepine CJ, Parker JO, Skopal J, Chumakova G, Kuch J, Wang W, Skettino SL, Wolff AA. Effects of ranolazine with atenolol, amlodipine, or diltiazem on exercise tolerance and angina frequency in patients with severe chronic angina: a randomized controlled trial. JAMA. 2004; 291: 309–316.
Antzelevitch C, Belardinelli L, Zygmunt AC, Burashnikov A, Di Diego JM, Fish JM, Cordeiro JM, Thomas G. Electrophysiological effects of ranolazine, a novel antianginal agent with antiarrhythmic properties. Circulation. 2004; 110: 904–910.
Wang P, Fraser H, Lloyd SG, McVeigh JJ, Belardinelli L, Chatham JC. A comparison between ranolazine and CVT-4325, a novel inhibitor of fatty acid oxidation, on cardiac metabolism and left ventricular function in rat isolated perfused heart during ischemia and reperfusion. J Pharmacol Exp Ther. 2007; 321: 213–220.
Undrovinas AI, Fleidervish IA, Makielski JC. Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine. Circ Res. 1992; 71: 1231–1241.
Hwang H, Arcidi JMJ, Hale SL, Simkhovich BZ, Belardinelli L, Dhalla AK, Shryock JC, Kloner RA. Ranolazine as an adjunct to cardioplegia: A potential new therapeutic application. J Cardiovasc Pharmacol Ther. 2009; 14: 125–133.
Bers DM, Barry WH, Despa S. Intracellular Na+ regulation in cardiac myocytes. Cardiovasc Res. 2003; 57: 897–912.
Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res. 2000; 87: 275–281.