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J Am Coll Cardiol, 2003; 41:1611-1622, doi:10.1016/S0735-1097(03)00244-4 © 2003 by the American College of Cardiology Foundation |
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* Division of Cardiology, Department of Internal Medicine, Cincinnati, Ohio, USA
Institute of Molecular Pharmacology and Biophysics, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
Manuscript received September 13, 2002; revised manuscript received December 9, 2002, accepted December 18, 2002.
* Reprint requests and correspondence: Dr. Arnold Schwartz, Institute of Molecular Pharmacology and Biophysics, Department of Surgery, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, Ohio, USA 45267-0828.
schwara{at}email.uc.edu
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BACKGROUND: Prolongation of APD and reduction in current density of Ito are thought to be hallmarks of VR and HF. Mechanisms are not understood.
METHODS: Patch-clamp, perfused hearts, echocardiography, and Western blots were employed using 2-, 4-, 8-, and 9- to 12-month-old Tg mice.
RESULTS: Transgenic mice developed slow VR statistically manifesting at four months and continuing through death at 12 to 14 months, despite a slight up-regulation of Ito. A slight decrease or no change in APD was observed up to eight months; however, at 9 to 12 months, a small increase in APD was detected. Early afterdepolarizations were observed after application of 4-aminopyridine in Tg mice. No change was detected in protein of Kv4.3 and Kv4.2 up to eight months. At 9 to 12 months, Tg mice showed a slight decrease (41.4 ± 6.9%, p < 0.05) in Kv4.2, consistent with a decrease in Ito. Surprisingly, Kv1.4 (the "fetal" K+-channel form) was up-regulated, and restitution of Ito was slowed. Echocardiography revealed cardiac enlargement with impaired chamber function in hearts that were taken from the older animals.
CONCLUSIONS: Contrary to accepted dogma, APD and Ito in a mouse model of hypertrophy and HF are not hallmarks of pathophysiology. We suggest that [Ca2+]i (i.e., [Ca2+] concentration) is the primary factor in triggering cardiac enlargement and arrhythmogenesis.
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(4), and Gq (5) develop cardiac hypertrophy and, eventually, heart failure (HF) as the animals age. The anatomic process of hypertrophy is generally referred to as "ventricular remodeling." The latter is frequently accompanied by arrhythmogenic changes known as "electrical remodeling." The latter typically is thought to include prolongation of action potential duration (APD), down-regulation of transient outward potassium current (Ito), and inward rectifier potassium current (IK1), decreased responsiveness to ß-adrenergic stimulation, and alterations of intracellular Ca2+ handling. Gwathmey et al. (6) were the first to show prolongation of the Ca2+ signal and APD in trabeculae from failing human myocardium. Despite compelling evidence to implicate that Ito is a key contributor in the repolarization process of the ventricular action potential (7), the role of Ito in the APD remains unclear. The changes in Ito that have been reported in hypertrophy and subsequent to myocardial infarction and HF are equivocal. Down-regulation of Ito has been described in a number of rat models of myocardial hypertrophy (8) and in human HF (9). In Tg mouse models of HF where Gq (5) or calsequestrin is over-expressed (1), a marked down-regulation of Ito density appears to be associated with a prolongation of the APD. On the other hand, there are reports of an "up-regulation" of Ito in hypertrophied cardiac myocytes (10–12) and in cardiac myocytes after induced myocardial infarction (13). Taken together, cellular hypertrophy is not always linked to a reduction in Ito. The complexities of K+-associated currents and the APD, as well as difficulties in trying to "link" Ito and APD, have recently been nicely discussed by Roden (14). On the other hand, most investigators, perhaps more intuitively than factually, feel that increases in intracellular Ca2+ concentration ([Ca2+]i) may lead to contractile dysfunction, hypertrophy, and HF (15) and that [Ca2+]i increases may trigger downstream signaling cascades that initiate a "hypertrophic gene program" (16). Enhanced Ca2+ entry can extend the plateau phase of the action potential, thereby increasing the APD that, in turn, would at least theoretically result in elevated [Ca2+]i, causing hypercontractility of an already compromised heart and arrhythmias at the onset of cardiac hypertrophy; Ca2+ entering through the L-type voltage-dependent calcium channel (L-VDCC) not only serves as a trigger for contraction but also may transduce the electrical signals into a series of biochemical signals in the heart. Backx et al. (17) supported this hypothesis in mice over-expressing an N-terminal fragment of Kv4.2, by showing that verapamil treatment prevented the progressively developed cardiomyopathy. The specific role of altered L-type Ca2+ channels in the impaired Ca2+ homeostasis of the failing heart (1,5,18,19) is an area of considerable controversy.
We generated Tg mice, over-expressing the cardiac 1 subunit of the L-VDCC (20) in order to simulate a scenario in which there would be a sustained but low-level of an increased intracellular calcium. These animals exhibited a slowly developing hypertrophy and eventually died with a cardiomyopathic HF phenotype (21). We hypothesize that a sustained increase of inward calcium current (ICa) (22,23), regardless of the source, is a major important mediator of hypertrophy associated with electrical remodeling. We studied these mice in order to reveal the underlying electrophysiologic basis of the electrical remodeling. Accordingly, the present study was designed to systematically characterize the electrophysiologic properties (Ito and APD) of cardiac myocytes derived during aging in these Tg mice in order to determine if there is a consistent pattern of ion current changes in both ventricular and electrical remodeling. In this study, we report in vivo, in situ, and cellular evidence that Ca2+ is the critical signal of cardiac hypertrophy and that the APD and Ito are hallmarks of the electrical, but not the ventricular, remodeling processes.
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Western blot analysis. Membrane fractions were prepared from mouse hearts as previously described (21). Protein samples were separated on SDS-PAGE, transferred to nitrocellulose membranes, and blocked in TBS-5% milk. Primary antibodies were incubated overnight at 4°C (anti-Kv1.4 ALOMONE, anti-Kv4.2, and Kv4.3; Chemicon Int.). Secondary antibodies were peroxidase-labeled anti-rabbit or anti-mouse IgG (Amersham). Antibody signals were detected by enhanced chemiluminescence and quantified by densitometry (Alpha Imager 2000, version 3.1; Alpha Innotech Corp.). All measurements were normalized to protein levels of mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon Int.).
Ex vivo hemodynamic measurements. The hearts were perfused in a retrograde manner at a constant coronary pressure (50 mm Hg) with oxygenated Krebs-Henseleit Buffer (37.4°C) in 9- to 12-month-old animals and in work-performing heart preparations in younger animals as previously described (23).
Echocardiography.
In vivo ventricular function was measured with a noninvasive technique as previously described (24). Noninvasive echocardiography was performed in 1-over-expressed Tg and Ntg mice at ages of four and eight months. Noninvasive cardiac function was assessed by two-dimensional guided M-mode echocardiography.
Statistical analysis. Comparison between gene expression levels were made using unpaired Student t test or two-way analysis of variance (followed by Bonferroni’s method for post-hoc pairwise multiple comparisons) for electrophysiology (EP) data to account for differences between individual animals and individual cells. Values are expressed as mean ± SEM and considered significantly different at p < 0.05. Samples sizes (n) are listed as n = x/y to denote x cells from y mice; only one measurement per cell was performed.
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Potassium currents. Subsequent experiments were designed to examine K+ currents. The IK1 is important in maintaining the resting membrane potential and in shaping the contour of the AP (Figs. 2A to 2D). We found no significant differences between the groups in IK1 current-voltage relationships up to nine months of age over a broad range of membrane potentials, particularly at the membrane potential range encountered during phase III repolarization of the AP, that is, from –40 mV to 80 mV. Despite the decrease in the density of IK1 after nine months in the Tg mice, the resting membrane potential was not significantly altered (Tables 1 and 2).
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We then investigated whether changes in the APD were associated with alterations in Ito, as has been suggested (7). The current-voltage relationships for Ito,peak and Ito are illustrated in Figures 3A and 3B. In both Ntg and Tg cells, the threshold for the activation of Ito was about –20 mV. The Ito in the Tg cells was significantly higher in all age groups except in the 9- to 12-month-old Tg compared with the Ntg cells (Table 2). Similar to Ito,peak density, the Isus, was also significantly higher than Ntg in the two-, four-, and eight-month Tg myocytes at membrane voltages of 30 to 80 mV. The Isus density remained the same as in the Ntg myocytes in the 9- to 12-month-old mice. It is conceivable that with progression to hypertrophy and HF, a decrease in the density of the Isus occurs (Figs. 3D to 3F, Table 2). Steady-state inactivation curves were obtained in the Ntg and Tg myocytes to compare the availability of Ito channels for activation and to determine whether the observed changes in current density are associated with altered channel kinetics. Under these conditions, the residual steady-state current was approximately 50% of the peak current level (Fig. 4A). The mean values of V0.5 and k for the Ntg and the Tg cells were evaluated in presence of 0.3 mM CdCl2. No significant difference was observed in V0.5 between the Ntg and Tg cells at 4- and 9- to 12-month age groups (Table 2). We previously considered whether changes in the availability of Ca2+ channels could account for the increased ICa (21,22); therefore, in addition, we analyzed the steady-state inactivation characteristics of ICa shown in Figure 4B and Table 2.
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fast) between the Ntg and Tg cells in the four-month-old age group (Ntg, 30.9 ± 1.2 ms, n = 27/4 vs. Tg, 36.2 ± 0.7 ms, n = 37/4, p < 0.05). The slow was not significantly different between Ntg and Tg mice at this age. In contrast, slow was significantly increased in myocytes from 9- to 12-month-old Tg mice at +40 mV (Ntg, 580.8 ± 80.3 ms, n=27/5, vs. Tg, 938.7 ± 123.5 ms, n = 30/5) (Figs. 4C and 4D). The slowed inactivation kinetics of Ito suggested differences in recovery from inactivation, prompting the study of restitution of Ito. The recovery from inactivation of Ito in Ntg and Tg mouse cardiac myocytes was displayed in Figures 4E and 4F, respectively. The slow at –80 mV is 237.9 ± 16.2 ms, n = 14/3, in the Ntg was significantly shorter than that in the Tg group, 491.2 ± 34.8 ms, n = 36/4, p < 0.001 (Table 2).
Western blot analysis was performed to see whether the changes in Ito density could be correlated with quantitative changes in the K+ channel protein expression. Interestingly, the Kv1.4 protein was up-regulated in the eight-month-old mice (Figs. 5A and 5B). The increase in Kv1.4 coincided with the increase of the slow of recovery from inactivation. In spite of this up-regulation, Kv4.3 protein levels were not altered in the two-, four-, and eight-month-old Tg mice (Fig. 5A, middle panel). Densitometric analysis of Kv4.2 from 9- to 12-month-old Tg mice revealed a decrease 41.4 ± 6.9% (n = 3, p < 0.05) compared with Ntg. This observation is consistent with the EP data on Ito (Fig. 3) indicating a 34% decrease.
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Xu et al. (27), in the dominant-negative Kv2 subunit mice, showed that IK,slow (slow component of the delayed rectifier current) was selectively reduced and was associated with a marked AP prolongation in ventricular myocytes; however, no signs of hypertrophy were observed. It is apparent that removal of Kv4- and Kv2-mediated currents (Ito) do not pari passu result in cardiac hypertrophy.
In contrast to a number of models of cardiac hypertrophy previously documented in which there is a down-regulation of Ito and IK1, we observed a modest, though surprising, increase in Ito,peak, Isus, and Ito up to eight months of age in our Tg model of cardiac hypertrophy. Furthermore, our data regarding Isus agree with previous results obtained from calsequestrin over-expressed Tg mice (1). A variety of factors have been suggested to account for this phenomenon, but the mechanisms are still unclear (28). We offer the speculation that the slight Ito up-regulation may provide a protecting "balance mechanism" to obtund excessive lengthening of the APD and Ca2+ flux, in order to minimize the incidence or onset of fatal ventricular arrhythmias.
Our data on Ito and ICa (21) provide important but opposing roles in the prolongation of the APD. The changes in Ito,peak and Isus (up to eight months of age) should contribute to a shortening of APD and, indeed, we observed perhaps a slight decrease or no change in APD in the two-, four-, and eight-month-old Tg cardiac myocytes. One must consider that the altered expression of Ca2+-handling proteins plays a significant role in prolongation of APD, and the relationship between APD and Ito,fast is multifactorial and complicated, at least according to model studies (29). Prolonged APD in human ventricular myocytes from patients with HF (30) may theoretically cause a slowed diastolic decline of [Ca2+]i and a reduced Ca2+ sequestration by the sarcoplasmic reticulum, thereby elevating diastolic Ca2+. In our experiments, the APD was slightly decreased in cardiac myocytes, recorded from four-month-old Tg mice. One possibility to account for this change is that an acceleration of [Ca2+]i reuptake occurs, which should increase the sarcoplasmic reticulum calcium load and, thus, augment systolic myocardial function (20,23). Indeed, this hypothesis was supported by Song et al. (22) who found an enhanced rate of Ca2+ transient decay in our four-month-old Tg mice. A logical explanation for the APD prolongation at the end stage of HF could be enhanced ICa (21,22), Na+-Ca2+ exchanger (NCX) (22) and down-regulation of Ito. Moreover, the protein expression level of Kv4.2 (underlying the cardiac Ito,fast) decreased at the advanced ages.
The NCX also plays an important role in Ca2+ homeostasis in mouse heart (31), and an increase in its forward mode of exchange might be expected to decrease contractility. We observed, however, an increased basal contractility in the four-month-old Tg mice, and the NCX activity was up-regulated (22). Less information is available concerning the reverse mode of exchange via the NCX, but we cannot rule out the role in "Ca2+ overload," which is thought to lead to arrhythmias.
In the fetal mouse ventricle, the contribution of the predominant Kv1.4 subunit to Ito,slow has been confirmed in Kv1.4 knockout mice (32), and in animals generated by crossbreeding the Kv4.2 (W362F) and the Kv1.4–/– mice (33). The fetal Kv1.4 subunit disappears in the adult, but we have now documented its "reappearance" during the hypertrophy process. Guo et al. (33) and Wickenden et al. (34) suggest that the up-regulation of Kv1.4 subunit (and Ito,slow) is a "protective mechanism" against arrhythmogenesis. We feel that such a process occurs in the present mouse model. We found a correlation between slow recovery kinetics of Ito and an elevation in Kv1.4 protein levels in the Tg model. We did not observe differences in the steady-state inactivation of Ito between Tg and Ntg; therefore, the slight up-regulation in Ito density in the Tg myocytes appears to be due to an increased number of functional K+ channels.
Our results also revealed an occurrence of EADs in phase 2 of the AP from eight-month-old Tg myocytes after application of the non-specific K+ channel blocker 4-AP. This is quite important. It has been suggested that L-type ICa and NCX current are candidate inward currents for initiating arrhythmia-triggering EAD in electrically remodeled myocardium (35); however, the precise molecular mechanism, responsible for the EAD generation, has not been defined and/or is controversial (36,37).
Study limitations. Gender differences and regional differences in current densities of myocytes are well known, but, at this time, we did not take these parameters into consideration.
Conclusions. Taken together, our electrophysiologic findings show that the sustained, increased ingress of Ca2+ (21,22) initiates a hypertrophy gene program in a very slow, but progressive manner leading eventually from hypertrophy to HF. We suggest that the progression of the disease is accompanied by electrophysiologic remodeling. To recapitulate, we observed an up-regulation of the Ito with no change in APD during the development of compensatory cardiac hypertrophy in the Tg mice up to eight months of age. Decreased Ito was associated with APD prolongation in the failing stage of cellular phenotypes with left ventricular dysfunction, from 9- to 12-month-old Tg mice.
These results can be considered as one case of partial uncoupling of electrophysiologic and structural remodeling in hypertrophy (38).
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protein in the hearts of transgenic mice. J Clin Invest. 1995;95:1676–1683[Medline]
q. Am J Physiol. 2000;279:H139–148
1 subunit of the L-type voltage-dependent Ca2+ channel in transgenic mice. J Biol Chem. 1999;274:21503–21506. Circulation. 2001;103:140–1471 subunit of L-type Ca2+ channel. Circ Res. 2002;90:174–181 subunit. Circ Res. 1998;83:560–567 subunit. Circ Res. 1999;85:623–633 subunit. Circ Res. 2000;87:73–79This article has been cited by other articles:
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