© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Differential effects of docosahexaenoic acid on contractions and L-type Ca2+ current in adult cardiac myocytes
aDepartment of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
bDepartment of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
gregory.ferrier{at}dal.ca
* Corresponding author. Tel.: +1-902-494-2550; fax: +1-902-494-1388
Received 20 July 2001; accepted 21 January 2002
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Beneficial effects of n–3 polyunsaturated fatty acids in Ca2+ overload have been attributed to blockade of L-type Ca2+ current (ICa-L). However, cardiac contractions may be maintained despite block of ICa-L. Objective: This study investigates the cellular basis by which docosahexaenoic acid (DHA), a representative n–3 polyunsaturated fatty acid, inhibits ICa-L while preserving contraction. Methods: Experiments were conducted in adult guinea pig ventricular myocytes with Na+ currents blocked. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and calcium-induced calcium release (CICR) triggered by ICa-L, were activated separately with voltage clamp techniques. Results: DHA (10 µM) inhibited ICa-L and CICR contractions but not VSRM contractions. CICR contractions exhibited a bell-shaped voltage-dependence. However, in the presence of DHA, only contractions with a sigmoidal voltage-dependence characteristic of the VSRM remained. These contractions exhibited inactivation properties characteristic of the VSRM. DHA abolished ICa-L elicited by test steps from –40 mV. Block was voltage-dependent, as residual ICa-L was elicited by steps from –70 mV. Cd2+ inhibited residual current, but not contractions initiated by the same activation steps. Conclusion: Preservation of VSRM contractions during block of ICa-L, may explain the ability of n–3 polyunsaturated fatty acids to inhibit Ca2+ influx while preserving cardiac contractile function.
KEYWORDS CICR calcium-induced calcium release; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ICa-L, L-type Ca2+ current; PUFA, polyunsaturated fatty acid(s); SR, sarcoplasmic reticulum; TTX, tetrodotoxin; Vh, half maximum steady-state inactivation voltage; VPC, postconditioning potential; VSRM, voltage-sensitive release mechanism
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Dietary n–3 polyunsaturated fatty acids (
-3 or n–3 PUFA, fish oils) decrease the risk of sudden cardiac death [1–4], which may reflect antiarrhythmic efficacy. Animals administered n–3 PUFA exhibit reduction in heart rate and ischemia-induced ventricular fibrillation [5,6]. n–3 PUFA also inhibit arrhythmic activity triggered by β-adrenergic agonists [7], lysophospholipids [8], ouabain [9,10] and elevated extracellular Ca2+ [11] in isolated cardiac myocytes. Two n–3 PUFA, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, have been shown to decrease electrical excitability and automaticity in cultured neonatal rat myocytes [12,13]. Antiarrhythmic effects of n–3 PUFA may be related to inhibition of Na+ [16,37] and/or Ca2+ currents [10,11,14–16]. DHA decreased peak L-type Ca2+ current (ICa-L) in rat ventricular myocytes [15,16], without significant changes in the voltage-dependence of activation or inactivation [15]. Inhibition of ICa-L by n–3 PUFA would be expected to inhibit contractions triggered by influx of Ca2+ and thereby decrease contractility. However, protective effects of n–3 PUFA in rat myocytes were not accompanied by depression of contraction [7,8,10]. The mechanism by which DHA preserves contractility while blocking ICa-L is investigated in the present study.
Thus far, effects of DHA on contraction have been considered only within the context of Ca2+-induced Ca2+ release (CICR). In CICR, Ca2+ entering cells primarily as ICa-L, binds to Ca2+ release channels (ryanodine receptors) in the sarcoplasmic reticulum (SR) and cause them to release SR Ca2+. The amount of Ca2+ released by CICR is graded by the amplitude of ICa-L [17]. Therefore, block of ICa-L by DHA should inhibit CICR.
Recent evidence indicates that cardiac excitation–contraction (EC) coupling also can be mediated by a second mechanism which is not graded by ICa-L [18–22]. The amplitudes of contractions and Ca2+ transients initiated by this mechanism show a sigmoidal dependence on membrane potential, rather than the bell-shaped proportionality to ICa-L exhibited by CICR [18,19,22]. Because this mechanism for Ca2+ release is graded by membrane potential, rather than magnitude of Ca2+ current, it is referred to as the voltage-sensitive release mechanism (VSRM) [18]. Contractions initiated by the VSRM and CICR can be activated separately and exhibit differential blockade with various ionic blockers and drugs [19–21,23–25]. For example, Cd2+ (100 µM) abolishes ICa-L and CICR with little effect on VSRM contractions [19,20,23–25]. Whether DHA affects initiation of contraction by the VSRM is not known.
It is possible that preservation of contractility during ICa-L block by n–3 PUFA might reflect differential effects on CICR and the VSRM. Therefore, the object of this study was to compare the effects of a representative n–3 PUFA, DHA, on the contribution of CICR and the VSRM to initiation of contractions in isolated cardiac myocytes.
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2 Methods |
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2.1 Myocyte isolation and experimental conditions
This study conforms to the Guide for the Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and guidelines published by the Canadian Council on Animal Care, and were approved by the Dalhousie University Committee on Animal Care. Ventricular myocytes were prepared as described in detail previously [18]. Adult male guinea pigs (350–450 g) were injected with heparin (3.3 i.u. g–1) and sodium pentobarbital (120 mg kg–1). Hearts were removed and perfused through the aorta with Ca2+- and Mg2+-free perfusate. Collagenase (0.5 mg/ml, Worthington I (202 U/mg) and protease (0.1 mg/ml; Sigma type XIV (5.2 U/mg) were then included in the perfusate for approximately 8 min. Subsequently the ventricles were minced and cells released by gentle agitation in a K+- and substrate-enriched solution. Myocytes were placed in a chamber on an inverted microscope, and were superfused at 3 ml min–1 with solution at 37 °C and containing (mM): 10 Hepes, 100 choline Cl, 55 NaCl, 4 KCl, 1 MgCl2, 2.5 CaCl2, 10 glucose (pH 7.4 with NaOH) [19,20]. Na+ current was blocked with lidocaine (200 µM) and/or tetrodotoxin (TTX, 50 µM). The Na+ concentration also was reduced to facilitate block of Na+ current. A stock solution of DHA (100 mM, in ethanol) was stored under N2 at –20 °C and diluted to 10 µM in buffer immediately prior to use [16,17]. The final concentration of ethanol (<0.05%) did not cause detectable changes in contractions or currents. Application of Cd2+ and Ni2+ to block Ca2+ currents was accomplished at 37 °C with a rapid solution changer. Blockers were applied after conditioning pulses, but 3 s in advance of test steps. Controls utilized the same timing, without blocker.
2.2 Measurement of ICa-L and contraction
Discontinuous single-electrode voltage clamp recordings (switching rate 8–12 kHz) were obtained with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA, USA). High resistance microelectrodes (18–22 M
) filled with 2.7 M KCl were used in most experiments, to minimize intracellular dialysis [19,23]. Other experiments utilized patch pipettes filled with (mM): 140 CsCl, 4 MgATP, 1 MgCl2, 0.12 CaCl2, 5.0–10.0 EGTA, 5 glucose, 0.05 8-bromo-cAMP, 10 Hepes, pH 7.2 with CsOH. pCLAMP software (v. 6.0) was used to generate voltage clamp protocols, and for data acquisition and analysis. Activation steps were preceded by a train of 10 conditioning pulses (200 ms at 2 Hz) from a holding potential of –80 to 0 mV, to provide a consistent history of activation. In many experiments, contractions initiated by the VSRM and CICR were activated separately by sequential steps from a post-conditioning potential (VPC) of –65 to –40 mV, and then to 0 mV, respectively [18,19,26]. Other protocols are described in the results and legends. ICa-L was measured as the difference between the peak inward current and a point at the end of the voltage step (200 ms). Contractions were measured as unloaded cell shortening with a video edge detector (Crescent Electronics, Sandy, UT, USA). Recordings were digitized with a Labmaster A/D interface (Axon Instruments, Foster City, CA, USA) and stored on hard disk.
2.3 Materials
DHA was obtained from Nu-Chek-Prep (Elysian, MN, USA) and was checked for purity and quantitated by GLC following conversion to its methyl ester [27]. TTX was purchased from Alomone Labs (Jerusalem, Israel) Lidocaine HCl, tetracaine and CdCl2 were purchased from Sigma–Aldrich Canada (Oakville, ON, Canada).
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3 Results |
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3.1 DHA selectively inhibits CICR
We first compared effects of DHA on VSRM- and CICR-induced contractions initiated by sequential activation steps to –40 and 0 mV from a VPC of –65 mV. In the absence of DHA (Fig. 1A), the step to –40 mV initiated a VSRM contraction with little inward current. The step to 0 mV elicited a CICR contraction and a large inward Ca2+ current, previously identified as ICa-L [18,19,23,24,26]. When myocytes were superfused with 10 µM DHA effects developed gradually. After 20 min, a large VSRM contraction still occurred with the step to –40 mV, but CICR contractions and ICa-L initiated by the step to 0 mV were virtually abolished (Fig. 1B). Fig. 2 shows that DHA did not significantly alter mean amplitudes of VSRM contractions, but dramatically reduced amplitudes of CICR-induced contractions by 93% (P<0.001) (Fig. 2, top). Mean data showed that DHA slightly but significantly (P<0.01) increased the small inward current observed with the step to –40 mV (Fig. 2, bottom). In contrast, the magnitude of peak ICa-L elicited by the step to 0 mV was strongly inhibited (approximately 85%) by DHA (P<0.001). Thus, our observations demonstrate that DHA selectively inhibits CICR, with little if any effect on VSRM contractions.
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3.2 Effects of DHA on contraction–voltage (CV) and current–voltage (IV) relations
To further investigate effects of DHA, we determined CV relations with activation steps to different membrane potentials. Effects of DHA on CICR in the absence of the VSRM were evaluated with test steps from a VPC of –40 mV to inactivate the VSRM [19] (Fig. 3A). Representative recordings of contractions and currents in the absence of DHA are shown in Fig. 3B. A step from –40 to 0 mV triggered inward ICa-L and a large contraction. When the test step was changed to +80 mV, inward current was no longer observed, and only a small phasic contraction occurred. Effects of a 15–20-min exposure to 10 µM DHA are shown in Fig. 3C. DHA abolished ICa-L and the phasic contraction elicited by the step to 0 mV in the control. The contraction initiated by the step to +80 mV also decreased in amplitude with DHA (Fig. 3C). Mean CV and IV relations are plotted in Fig. 4A and B, respectively. Before exposure to DHA, the CV curve was bell-shaped and closely reflected the profile of the IV relation for ICa-L, as expected for CICR [17]. Exposure to 10 µM DHA, strongly inhibited ICa-L and abolished CICR contractions at all activation steps (Fig. 4).
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IV and CV curves also were determined with a VPC of –70 mV (Fig. 5A). With this VPC, both CICR and the VSRM are available and may contribute to contraction [18,19,23,24,26]. Fig. 5B shows representative recordings before exposure to DHA. When the VPC was –70 mV, a phasic contraction was initiated by the step to –40 mV although little or no inward current was evident. With a step to 0 mV, a larger contraction and inward ICa-L were elicited. A large phasic contraction also was elicited at +80 mV, although inward current was no longer elicited at this potential. Fig. 5C shows corresponding records after the myocyte had been exposed to DHA. When the VPC was –70 mV, large, phasic contractions were elicited with all three test steps. ICa-L was clearly inhibited on the step to 0 mV, and remained absent at +80 mV. In this example, a small inward current was observed with the step to –40 mV.
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Mean CV and IV relationships determined with a VPC of –70 mV are illustrated in Fig. 6. In the absence of DHA the CV relationship was sigmoidal, as expected when the VSRM is available for activation [18,19,23,24,26]. Thus, contraction no longer reflected the bell-shaped IV relation. When DHA was included in the superfusate, the CV relation remained sigmoidal. Contractions exhibited a threshold near –60 mV, reached maximum amplitude near –20 mV, and remained maximal up to +80 mV. The plateau of the CV relation was somewhat lower and distinctly flatter than control. When the VPC was –70 mV, DHA decreased but did not abolish inward current (Fig. 6B). Current remaining in the presence of DHA exhibited a bell-shaped IV relation, with a peak near –20 mV. Inward current approached 0 nA near +40 mV. Thus, contraction clearly was not proportional to the current remaining in the presence of DHA. In these experiments, mean current decreased relative to control at all potentials. This differs from Fig. 2 where an increase was observed at –40 mV. This may reflect differences in protocols. CV and IV protocols utilized shorter VPC (500 vs. 4000 ms) and more repetitions of conditioning pulse trains.
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3.3 Are contractions remaining in the presence of DHA initiated by inward current?
DHA inhibited virtually all inward current elicited from a VPC of –40 mV, but current was still observed with a VPC of –70 mV (Figs. 4 and 6
). The protocols used are the same as those for separating L- and T-type Ca2+ currents. Thus it is possible that the current remaining with DHA is T-type Ca2+ current [28]. Indeed, with DHA, the IV relation shifted negatively, as expected if the current remaining was T-current. However, these results also might be explained if block of L-current is voltage dependent. To differentiate between these possibilities, we compared the effects of Ni2+, which preferentially blocks T-current, and Cd2+, which preferentially blocks ICa-L, on inward current remaining with DHA. TTX was used to eliminate Na+ current. Ni2+ or Cd2+ were applied with a rapid solution switcher 3 s before test steps. Fig. 7 shows that 200 µM Ni2+ reduced maximum peak inward current approximately 45%. However, application of 100 µM Cd2+ almost completely inhibited this inward current.
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The sensitivity of current to Cd2+ suggests that current remaining in the presence of DHA is L-type current. The negative IV relation in the presence of DHA might be caused by inward Na–Ca exchange current, which would be greatest at negative potentials. To eliminate this possibility we evaluated effects of DHA with patch pipettes containing 5–10 mM EGTA, to buffer changes in free intracellular Ca2+ caused by SR release. Under these conditions contractions were absent. Fig. 8A shows records of currents elicited with the two-step protocol with 2 mM (left) and 0 mM extracellular Ca2+ (right). These records also are shown superimposed to demonstrate that significant inward Ca2+ current was not elicited by a step –40 mV, and that ICa-L decayed completely within the second 200 ms step to 0 mV. Thus, measurement of peak inward current relative to net current at the end of the step accurately represents inward Ca2+ current at both potentials. Fig. 8B shows representative recordings used to determine IV relations. DHA strongly inhibited inward current elicited by a step from –40 to 0 mV (left). However, with a step from –70 to 0 mV, a larger current remained in the presence of DHA (right). Fig. 8C shows mean IV relations. DHA almost completely blocked inward Ca2+ current when steps were initiated from –40 mV. However, when steps were made from –70 mV inward current was blocked less. DHA reduced mean inward current at 0 mV to 0.20±0.07 nA (13.4%) with steps from –40 mV, but to 0.60±0.10 nA (26.8%) when steps were made from –70 mV (P<0.05). The negative shift of IV relations by DHA was virtually eliminated. Thus, effects of DHA are most likely explained by voltage-dependent block of ICa-L.
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To evaluate the role of residual ICa-L in initiation of contraction, we also examined the effects of Cd2+ on contraction amplitude. Because repeated applications of Cd2+ gradually caused spontaneous contractions, complete CV relations were not generated. Rather, effects were determined only for steps that elicited near maximal current. A representative example is shown in Fig. 9. In the presence of 10 µM DHA, a step to –10 mV elicited an inward current plus a contraction (Fig. 9A). Rapid application of 100 µM Cd2+ blocked the current; however, the amplitude of the contraction was not affected (Fig. 9B). Similar results were obtained in five experiments. Thus, contractions were independent of inward current, even with test steps to potentials at which current could be elicited in the presence of DHA.
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3.4 Steady-state inactivation of contractions in the presence of DHA
Since contractions in the presence of DHA were not proportional to ICa-L and could be elicited when current was blocked by Cd2+, it is likely that these contractions were initiated by the VSRM [18,19,23,26]. If this is correct, these contractions should show steady-state inactivation characteristic of the VSRM [19,23]. Steady-state inactivation curves were determined before and after exposure to 10 µM DHA, with the protocol illustrated in Fig. 10A. Contractions were elicited by an activation step to –40 mV. The activation step was preceded by a 700 ms long VPC. The VPC voltage was changed systematically in 5 mV steps with each repetition of the protocol. Immediately prior to the activation step the VPC was returned to –70 mV for 3 ms. Fig. 10 shows representative recordings of contractions (left) and currents (right) recorded in the presence of 10 µM DHA. Little if any contraction was elicited when the VPC was –30 mV. However, contractions increased with progressively more negative VPC, and became maximal with a VPC of –70 mV. Small inward currents were observed as well at VPC negative to –50 mV (Fig. 10, right).
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Fig. 11 shows mean inactivation curves for contraction in the absence and presence of DHA. In Fig. 11A, mean amplitudes of contractions are plotted as a function of VPC potential. The amplitudes of contractions before and after exposure to DHA were maximal at VPC equal or negative to –70 mV. The maximum amplitude of contraction was not affected by DHA. Contractions were negligible when the VPC was –30 mV, before and after exposure to DHA. The steady-state inactivation curves in Fig. 11B were normalized to the maximum contraction amplitude for each experiment. These data were fit to Boltzmann functions represented by the solid lines. In the presence of DHA, the half-inactivation voltage (Vh) was slightly more negative (–52.1 mV) than that for control myocytes before exposure to DHA (–46.0 mV). However, Vh after exposure to DHA was very similar to values previously reported for the VSRM [19,23], and to the value observed for the VSRM in the absence of DHA in the present study.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Our study demonstrates for the first time that a major n–3 PUFA, DHA, inhibits ICa-L, while preserving contraction in the same myocytes. Our study explains these apparently contradictory observations by demonstrating that DHA inhibits CICR by inhibiting its primary trigger, ICa-L, but does not inhibit the VSRM. Thus, DHA inhibits only one of two mechanisms for cardiac EC coupling.
4.1 Mechanism of EC-coupling in the presence of DHA
Earlier studies reported that inhibition of ICa-L by n–3 PUFA also decreased myocyte shortening [11] and Ca2+ transients [15]. These studies were conducted at low temperatures (22–32 °C) and with cells dialyzed with patch pipette solutions containing neither cAMP nor calmodulin. Either of these conditions would inhibit the VSRM [22,26]. We also found that DHA abolished contractions along with ICa-L when the VSRM was unavailable. However, contractions persisted when the VPC was –70 mV and both the VSRM and CICR were available. This can be explained by differential effects of DHA on the VSRM and CICR, as shown in experiments with sequential activation steps to –40 and 0 mV. DHA clearly inhibited CICR contractions but had little effect on contractions initiated by the VSRM.
The characteristics of contractions remaining in the presence of DHA indicate that they were initiated by the VSRM rather than CICR. Firstly, DHA abolished ICa-L and the bell-shaped CV curve determined from a VPC of –40 mV. However, contractions still could be initiated by steps from a VPC of –70 mV. These contractions exhibited a sigmoidal CV relation, in which amplitudes of contraction were independent of magnitude of inward current, which is typical of the VSRM [18,19,22]. Comparison of CV curves before and after DHA also suggest that DHA inhibited a bell-shaped component corresponding to CICR. Secondly, contractions in the presence of DHA were not inhibited by 100 µM Cd2+, which abolishes CICR [20,24]. Finally, our experiments demonstrate that the phasic contractions remaining in the presence of DHA exhibit a steady-state inactivation profile with a half-inactivation voltage (Vh) near –50 mV, which is characteristic of the VSRM, but approximately –25 mV negative to Vh for ICa-L [19,23].
It also is unlikely that contractions in the presence of DHA were initiated by CICR coupled to Ca2+ influx via routes other than ICa-L. TTX did not inhibit contractions elicited in the presence of DHA. Thus, these contractions cannot be attributed to Ca2+ influx through Na+ channels or to reverse mode Na+–Ca2+ exchange triggered by Na+ influx. It also is unlikely that these contractions were caused by reverse mode Na+–Ca2+ exchange, because they exhibited steady-state inactivation and could not be elicited from a VPC of –40 mV. Na+–Ca2+ exchange does not inactivate and has been reported to initiate contractions when the conditioning or holding potential is –40 mV [31,32].
4.2 Effects of DHA on Ca2+ current
DHA inhibited current more completely when the VPC was –40 rather than –70 mV. Xiao et al. observed a similar effect with EPA and suggested this represented voltage-dependent block of ICa-L [15]. However, it also is possible that current at negative potentials was T-type Ca2+ current [29], and that T-current might be resistant to n–3 PUFA. T-current is expected to be abolished by 200 µM Ni2+, but only slightly inhibited by 100 µM Cd2+ [28]. However, we found the reverse, which suggests the current is ICa-L [28].
DHA shifted the IV relation negatively as expected for T-current [29,30]. However, this likely reflects inward Na+–Ca2+ exchange current driven by Ca2+ transients at negative membrane potentials. The contribution of exchanger current would appear relatively greater when ICa-L is partially inhibited. This is supported by the absence of a negative shift when free intracellular Ca2+ was buffered with EGTA. Thus, our observations suggest that DHA causes voltage-dependent block of L-type Ca2+ current, and support the conclusions of Xiao et al. [15].
DHA stimulated a small inward current at negative potentials when protocols with very long VPC were used (Fig. 2). As this was not observed when intracellular Ca2+ was buffered, these inward currents may represent Na+–Ca2+ exchange. With protocols utilizing long VPC it is likely that use-dependent block of Na+ current by lidocaine is not complete during conditioning pulses from –80 mV. It is possible that DHA promoted exchange current by providing additional block of Na+ current [16,37] and reducing intracellular Na+ levels. This effect of DHA might be eliminated with protocols for IV relations that would maximize use-dependent block by lidocaine.
4.3 Selective block of CICR by DHA
Our observations indicate that DHA inhibits CICR by blocking ICa-L. In contrast, the VSRM was little affected by DHA and could be activated at potentials where ICa-L was minimal or when ICa-L was blocked by Cd2+. These observations could explain earlier studies in which n–3 PUFA reversed or prevented Ca2+ overload without inhibiting contraction [9,10], despite blockade of ICa-L [15,16]. Thus differential actions on CICR and the VSRM may allow n–3 PUFA to exert protective effects by reducing Ca2+ influx through ICa-L, while preserving myocardial function.
Time for primary review 34 days.
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Acknowledgements |
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The authors thank J.Q. Zhu and C. Guyette for excellent technical assistance. This study was supported in part by grants from the Heart and Stroke Foundations of Nova Scotia and New Brunswick, and the Medical Research Foundation of Canada.
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