Transmural Heterogeneity of Na+–Ca2+ Exchange
Evidence for Differential Expression in Normal and Failing Hearts
Spatial electrical heterogeneity has a profound effect on normal cardiac electrophysiology and genesis of cardiac arrhythmias in diseased hearts. The Na+–Ca2+ exchanger (NCX) is a key linker, through Ca2+ signaling, between contractility and arrhythmias. Here we characterize the differential transmural expression of NCX in normal and rapid pacing-induced failing canine hearts. Significant transmural heterogeneity of NCX was present in normal hearts, as NCX current density measured at +80 mV was significantly (P<0.05) greater in epicardial (EPI) (5.49 pA/pF) than mid-myocardial (MID) (2.84 pA/pF) and endocardial (ENDO) (2.21 pA/pF) cells. Interestingly, heart failure caused a selective increase in NCX current density (P<0.05) limited to ENDO (by 202%) and MID (by 76%) but not EPI myocytes (P=not significant). The differences in functional expression were associated with changes in both mRNA and protein levels. The normal EPI layer exhibited the greatest NCX mRNA and protein levels compared with MID and ENDO layers, whereas the ENDO layer underwent the most pronounced increase in mRNA (by 185%) and protein (by 207%) levels in heart failure. The transmural NCX gradient, from EPI (greatest) to ENDO (least), is disrupted in heart failure. A selective upregulation of NCX expression in MID and ENDO in heart failure markedly redirects the orientation of the transmural functional gradient of NCX and may lead to enhanced vulnerability to cardiac arrhythmias.
Sudden death is prevalent in patients with heart failure (HF).1 Abnormalities in functional expression of calcium-handling proteins are prominent in HF and constitute a crucial link between arrhythmias and decreased contraction. The Na+–Ca2+ exchanger (NCX) plays important roles in both contractile dysfunction and arrhythmogenesis in HF.2
Prolongation of action potential duration (APD) is a hallmark of failing myocytes, which is arrhythmogenic and contributes to the increased incidence of sudden death in patients with HF. More importantly, transmural dispersion of repolarization (TDR) has a crucial role in arrhythmogenesis.1 In failing hearts, downregulation of K+ currents and upregulation of NCX have been reported (see review by Tomaselli and Zipes1). The transmural homogenenous downregulation of K+ currents3 does not suffice to account for the exaggerated TDR characteristic of HF. We characterized the differential transmural expression of NCX in control and failing hearts. The alterations in transmural NCX heterogeneity in failing hearts highlight a potential substrate for the production of lethal arrhythmias in HF.
Materials and Methods
Real-time quantitative RT-PCR, Western blotting, and whole-cell patch clamp were used to measure the NCX mRNA, immunoreactive protein levels, and current, respectively. An expanded Materials and Methods can be found in the online data supplement available at http://circres.ahajournals.org.
Figure 1A illustrates representative traces of Ni2+-sensitive current in a normal MID cell. To confirm that the Ni2+-sensitive current is NCX current (INCX), substitution of extracellular Na+ with Li+ eliminated the current (online Figure I). Figure 1B shows representative INCX traces recorded in myocytes isolated from epicardial (EPI), mid-myocardial (MID), and endocardial (ENDO) layers. The magnitude of INCX, both inward and outward, was the largest in EPI cells (P<0.05). In HF, the INCX density was significantly increased in MID (Figure 1D) and ENDO (Figure 1F) (P<0.05) but not in EPI cells (Figure 1E). The current density measured at −100 mV and +80 mV in normal and failing myocytes were summarized in Figure 2A and 2B. Significant transmural heterogeneity of INCX was found in normal hearts with the largest current density in EPI (5.49 pA/pF, +80 mV) cells. HF selectively enhanced NCX functional expression in ENDO and MID myocytes (Figure 2B). The largest increase in the ENDO (202%) and significant increase in the MID (76%) layers disrupted the orientation of the basal transmural INCX gradient.
Real-time quantitative RT-PCR was used to quantify the mRNA levels. The EPI layer exhibited the largest mRNA expression among three layers of normal hearts (Figure 3A). In HF, mRNA expression was markedly increased in ENDO and MID but not in the EPI layer. The largest increase in HF was found in the ENDO layer (Figure 3B). The real-time PCR results were consistent with the HF-associated change in INCX. Figure 3C shows a representative Western blot demonstrating different gradients of NCX immunoreactive protein in normal and failing hearts. In normal hearts, the NCX protein level was the greatest in the EPI layer, whereas it was selectively increased in the ENDO and MID layers of failing hearts (Figure 3C through 3E), disrupting the normal transmural protein gradient of NCX.
There are few reports concerning the cardiac transmural NCX heterogeneity, and the results are at variance.4–6 Consistent with our study, 1 report showed the INCX density in normal canine MID and EPI layers was larger than that in the ENDO layer; however, no data were provided for the mRNA or protein expression.4 In contrast, another study did not detect the difference in NCX protein expression between EPI and ENDO layers in normal canine hearts and attributed more rapid calcium transient decay in the EPI (versus ENDO) layer to increased expression of the sarcoplasmic reticulum Ca2+–ATPase (SERCA).5 However, only a 70-kDa proteolytic fragment of NCX was quantified, and no direct measurements of INCX were performed.5 In a rabbit model of myocardial infarction, ENDO cells exhibited greater NCX expression and function than EPI cells in both control and failing hearts.6 In humans, Northern blot analysis showed no difference in NCX mRNA levels between EPI and ENDO in both normal and failing hearts, but the authors noted large interindividual variability of NCX mRNA levels.7 Furthermore, Northern blotting is a less-robust mRNA quantification method than real-time quantitative PCR. Taken together, the differences in methodology, species, and HF models may contribute to discrepancies among different studies. Here we showed that the EPI layer consistently had greater NCX mRNA, protein expression, and current density than ENDO in normal hearts. Given the predominant forward mode of NCX, the higher INCX density in normal EPI cells is likely to be associated with greater Ca2+ entry into EPI than ENDO myocytes.8,9 Defective SERCA function may impair contractile performance in HF. In human HF, there is a transmural gradient of SERCA2a, decreasing from EPI to ENDO.7 It is tempting to speculate that the most profound increase in NCX in the canine failing ENDO layer may compensate for reduced SERCA2a expression.
The exaggerated transmural heterogeneity of electrophysiological and Ca2+-handling properties in HF may play an important role in arrhythmogenesis. Despite an increase in the transmural dispersion of APD,10 transmural K+ currents are homogenously reduced and L-type Ca2+ current is unaltered in rapid pacing-induced canine HF.3 In contrast, our data demonstrated selective enhancement of NCX in the MID and ENDO layers. The impact of disrupted transmural NCX heterogeneity on cardiac arrhythmogenesis is likely to be manifold, including the induction of triggered activity and promotion of functional reentry.
Triggered activity is a major mechanism for initiating arrhythmias in nonischemic HF. In rabbit nonischemic HF, spontaneous ventricular tachycardia has been attributed to subendocardial nonreentrant activation,11 and delayed afterdepolarizations (DADs) were observed.12 In human HF, the transient inward current, Iti, and DADs exclusively result from INCX.13 Furthermore, ventricular arrhythmias in patients with end-stage idiopathic cardiomyopathy primarily occur in the subendocardium by a focal nonreentrant mechanism, probably resulting from EADs or DADs.14 Our findings of a pronounced NCX increase in ENDO layers of nonischemic cardiomyopathic hearts fit well with these observations.11,12,14 INCX also plays a role in the genesis of EADs.15 Inward INCX may prolong the APD and set the stage for EADs as well as the associated TDR that favors functional reentry. Indeed, EADs,3 increased spatial dispersion of monophasic APD,16 and sudden death16 were observed in this canine HF model.
In summary, the normal transmural NCX gradient, oriented from EPI (greatest) to ENDO (least), is disrupted in HF. Selective upregulation in NCX expression in ENDO and MID with the greatest increase in ENDO markedly redirects the orientation of the basal transmural gradient of NCX. This may lead to enhanced vulnerability to cardiac arrhythmias in HF. Therefore, the present study sheds new light on transmural heterogeneity of NCX and potential substrates for cardiac arrhythmias, although the precise nature of its pathophysiological consequences such as exaggerated TDR, EADs, and DADs warrants further investigation.
The Ni2+-sentitive current was taken as INCX. Although Ni2+ is not a specific blocker of NCX, other channels and transporters were inhibited. Moreover, we validated that there was virtually no difference between external Na+- and Ca2+-induced INCX and Ni2+-sensitive currents in the present study.
This work was supported by the NIH grants P50HL52307 (to G.F.T.) and PO1HL077180 (to G.F.T.) and American Heart Association Postdoctoral Fellowship Grant 0225589U (to W.X.).
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received May 3, 2005; revision received June 16, 2005; accepted June 23, 2005.
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