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Changes in ionic currents and reduced conduction velocity in hypertrophied ventricular myocardium of Xin[alpha]-deficient mice.

ABSTRACT

Objective: mXin[alpha], a downstream target gene of Nkx2.5 transcription factor, was shown to encode a proline-rich and Xin repeats-containing protein which localizes to the intercalated disc of adult hearts. Our previous voltage-clamp studies have shown that the ventricular myocytes of mXin[alpha]-deficient mice exhibited a significant reduction in [K.sup.+] currents (Ito and IK1), L-type [Ca.sup.2+] currents, and maximum diastolic potential, leading to the development of early afterdepolarization (EAD) and arrhythmias. However, changes in cationic inward currents could also contribute to the genesis of EAD and arrhythmias in mXin[alpha]-deficient mice.

Methods: The present study aims to characterize changes in [Na.sup.+] currents on depolarization and transient inward currents (Iti) on repolarization. Conduction velocity (CV) on the frontal surface of ventricles were also measured and compared.

Results: Results of optical mapping on the Langendorff-perfused hearts at 37[degrees]C revealed a 36% reduction of CV in mXin[alpha]-/- ventricle. Pacing (3 Hz)-induced tachyarrhythmias were more frequently found and ventricular fibrillation (VF, 21 Hz for 5 min) occurred in one out of 8 mXin[alpha]-/- heart. When perfused at 30[degrees]C, no VF was observed in both types of preparations. Voltage-clamp study on isolated ventricular myocytes at 37[degrees]C shows increase in INa and Iti in mXin[alpha]-/- cardiomyocytes thus could explain the occurrence of re-entrant triggered arrhythmias.

Conclusion: The present results revealed that the CV was slower, but INa and Its were increased in mXin[alpha]-/-cardiomyocytes thus were prone to reentrant triggered arrhythmias. Hypothermia could reduce the occurrence of arrhythmias.

Keywords: arrhythmia, conduction velocity, ionic currents, Xin[alpha]-deficient murine heart, temperature, voltage-clamp studies

Introduction

Ablation of mXin[alpha] gene, encoding an intercalated discs protein (1), results in cardiac hypertrophy and cardiomyopathy with a reduction in connexin 43 expression (2). Previous voltage clamp study revealed that mXin[alpha]-deficient ventricular myocytes have markedly reduced L-type [Ca.sup.2+] currents, transient outward [K.sup.+] currents and barium-sensitive inward rectifier [K.sup.+] currents, in agreement with some of the abnormal action potential (AP) characteristics, such as prolonged action potential (AP) and reduced maximal diastolic potential (3). However, further experiments are required to explain the ionic mechanisms, in addition to the reduced [K.sup.+] currents, leading to the development of early afterdepolarizations (EADs) and slow response APs (4) observed in mXin[alpha]-/- myocytes. Changes in inward cationic currents might also contribute to EADs, slow response APs during depolarization and transient inward currents (Iti) on repolarization (5). The present study aims to characterize changes in [Na.sup.+] currents, Iti and conduction velocity (CV) of the mXin[alpha]-/- murine ventricular myocardium.

Methods

Whole-cell patch-clamp techniques (6, 7) were used on ventricular myocytes isolated enzymatically from 16 to 20-week-old wild-type and mXin[alpha]-/- male mice. Inward [Na.sup.+] currents were measured on depolarization for 40 ms in 22-24[degrees]C Tyrode solution containing 5 mM NaCl (133 mM NaCl was replaced by equimolar CsCl), 5 mM HEPES and 2 [micro]M nifedipine (an ICa,L blocker). Transient inward currents (Iti) were measured on repolarization after a series of depolarizing pulses (50-3050 ms) in 37[degrees]C HEPES-Tyrode solution as described (5). Optical mapping technique was used to measure CV of murine ventricular myocardium as described previously (8). In brief, the ascending aorta was cannulated and the heart was perfused with standard oxygenated Tyrode solution (2.5 ml/min) of the following composition (in mM): NaCl 137, NaHCO3 15.5, NaH2PO4 0.7, CaCl2 1.8, KCl 4, MgCl2 1, and glucose 11.1, gassed with gas mixture (95% 02-5% C02) to pH 7.2-7.4 (37[degrees]C). Murine heart was stained with 15 mL standard oxygenated Tyrode solution plus 30 [micro]L of 2 mM di-4-ANEPPS in DMSO (Molecular Probe, Paisley, UK) to monitor cardiac APs and paced (3 Hz, 3 ms in duration, 3 fold threshold voltage) at the basal portion of the right ventricle from a stimulator (S88J, Grass, West Warwick, Rhode Island, USA). The murine heart was further perfused with 100 mL standard oxygenated Tyrode solu tion plus 25 [micor]L of 10 [micro]M cytochalasin D (Sigma, Missouri, USA) to block cardiac contraction. The APs were then recorded and the CV was calculated as described (8) on the frontal surface of ventricle using optical mapping method.

Statistical Analysis

Quantitative data were expressed as mean [+ or -] S.E.M. Unpaired Student's t test and Chi-square test were used for comparison of data between groups. P values less than 0.05 were considered to be statistically significant.

Results

Measurement of CV on the frontal surface of ventricle in Langendorff perfused heart

Figure 1 shows representative activation maps obtained from the frontal surface of ventricles of mXin[alpha]+/+ and mXin[alpha]-/ hearts. When paced at the base portion of the right ventricle, activation spreads progressively in the mXin[alpha]+/+ heart. In contrast, a slower activation with frequently interrupted propagation was observed in the mXin[alpha]-/- heart. The CV could be determined from these activation maps. The CV was significantly slower in mXin[alpha]-/- heart than in mXin[alpha]+/+ heart. At 37[degrees]C, the average CV calculated from 11 mXin[alpha]+/+ ventricles was 86 [+ or -] 9 cm/s, whereas the CV from 6 mXin[alpha]-/- ventricles reduced significantly to 55 [+ or -] 7 cm/s (p<0.05). The incidence of pacing (3 Hz) induced ventricular tachycardia (VT) was 1 in 12 mXin[alpha]+/+ preparations, whereas the frequency (4 out of 8) of pacing induced VT was significantly higher in mXin[alpha]-/- preparations (p<0.05). Ventricular fibrillation (VF, 21 Hz for 5 min) occurred in one out of 8 mXin[alpha]-/- ventricles but none in 12 mXin[alpha]+/+ ventricles (1/8 vs. 0/12, p>0.05). At a lower temperature (30[degrees]C), the CV of 3 mXin[alpha]+/+ ventricles decreased to 60 [+ or -] 14 cm/s and, interestingly, there was no significant changes in the CV of 3 mXin[alpha]-/- ventricles (56 [+ or -] 8 cm/s). At 30[degrees]C no VT was observed in both groups of ventricles although the incidence of VT appeared higher in mXin[alpha]-/- (4/5) ventricles as compared to 1/4 in mXin[alpha]+/+ ventricles (p>0.05). Thus, a moderate hypothermia appears to reduce the occurrence of arrhythmias.

[FIGURE 1 OMITTED]

Experiments on ventricular myocytes isolated from wild type and mXin[alpha]-/- mice

The membrane capacitance (Cm) of isolated ventricular myocytes were measured by a small hyperpolarizing step (from -50 to -55 mV) (5-7). Results revealed a 38% larger Cm (and therefore larger surface area) in mXin[alpha]-/- than in mXin[alpha]+/+ ventricular myocytes, consistent with hypertrophied mXin[alpha]-/ myocytes detected previously (2).

We determined further the [Na.sup.+] inward currents (INa) in ventricular myocytes perfused in 22-24[degrees]C solution. Extracellular [[Na.sup.+]o was reduced to 5 mM in the presence of 133 mM [Cs.sup.+], 5 mM HEPES and 2 [micro]M nifedipine. As shown in Figure 2, INa inward currents on depolarization was significantly larger in mXin[alpha]-/- than in mXin[alpha]+/+ ventricular myocyte. In 37[degrees]C HEPES-Tyrode solution (containing 137 mM [[Na.sup.+]o), on repolarization to the holding potential (-40 mV) after prolonged depolarizing pulses (for 1050-3050 ms) (see Figure 3), the average oscillatory transient inward currents (Iti) after depolarizing pulse of 3050 ms (5) was 0.488 [+ or -] 0.081 pA/pF for 28 mXin[alpha]-/- ventricular myocytes. For 27 mXin[alpha]+/+ myocytes, similar clamp protocol induced significantly smaller Its (average values 0.203 [+ or -] 0.059 pA/pF, p<0.05). Also average Its in the 27 mXin[alpha]+/- (0.508 [+ or -] 0.103 pA/pF) were significantly larger than the 27 mXin[alpha]+/+ myocytes (p<0.05).

[FIGURES 2-3 OMITTED]

Results of these patch-clamp studies indicate that both INa currents induced on depolarization and the arrhythmogenic Iti currents generated on repolarization to the holding potential were increased in mXin[alpha]-/- and mXin[alpha]+/- hearts.

Discussion

The present results, together with our previous report (3), clearly show that mXin[alpha]-/- mouse myocytes exhibit increased Na' inward currents, reduced transient outward K, currents and inwardly rectifying [K.sup.+], currents in concomitant with prolonged AP and slower CV. These changes in electrophysiological properties of ventricular myocytes likely render mice being prone to develop EAD and VF. The enhanced Its in mXin[alpha]-/- ventricular myocytes is in contrast to the depressed Its observed in ventricular myocytes of dilated myopathic Syrian hamster (5). Thus the underlying cellular mechanisms for hypertrophied mXin[alpha]-/- myocardium (2, 9) were different from that of dilated cardiomyopathic hamster model (5, 10). We will incorporate research techniques used in the laboratory of Roshchevsky (11) in our joint research works in the future.

Acknowledgements

The present studies were supported by grants NSC95-2320 B016-013 (CIL), NSC95-2923-BO16-001-MY2 (CIL, YCC), NSC-RFBR 050490586 (MPR) from National Science Council, MMH-95103 from the Mackay Memorial Hospital, Taipei, Taiwan, R.O.C., and a grant R01 HLO75015 from the NIH (JJCL), U.S.A.

References

(1.) Wang DZ, Reiter RS, Lin JL, Wang Q, Williams HS, Krob SL, et al. Requirement of a novel gene, Xin, in cardiac morphogenesis. Development 1999;126: 1281-94.

(2.) Gustafson-Wagner EA, Lin JLC, Sinn H, Wang DZ, Reiter RS, Yang B, et al. Targeted deletion of mXin[alpha] gene, encoding an intercalated disc protein, leads to cardiac hypertrophy (Abstract). AHA Scientific Sessions 2006; 2006 Nov 12-15; Chicago, IL, USA; Circulation 2006;114 (Suppl): II-54.

(3.) Cheng CP, Loh YX, Lin CI, Lai YJ, Chen YC, Sytwu HK, et al. Electrophysiological characteristics of ventricular myocytes of Xina-deficient mice. In: Kimchi A, editor. Advances in Heart Disease. Proceedings of the 12th World Congress on Heart Disease; 2005 July 16-19; Vancouver, Canada; s.r.l. Bolongna, Italy; MEDIMOND: 2005. p. 25-9.

(4.) Bers DM. Cardiac excitation-contraction coupling. Nature 2002; 415: 198-205.

(5.) Wu SH, Chen YC, Higa S, Lin CI. Oscillatory transient inward currents in ventricular myocytes of healthy versus myopathic Syrian hamster. Clin Exp Pharmacol Physiol 2004; 31: 668-76.

(6.) Chen YJ, Chen SA, Chen YC, Yeh HI, Chan P, Chang MS, et al. Rapid atrial pacing increases the arrhythmogenic activity of single cardiomyocytes from pulmonary veins: implication in initiation of atrial fibrillation. Circulation 2001;104: 2849-54.

(7.) Chen YJ, Chen SA, Chen YC, Yeh HI, Chang MS, Lin CI. Electrophysiology of single cardiomyocytes isolated from rabbit pulmonary veins: implication in initiation of focal atrial fibrillation. Basic Res Cardiol 2002; 97: 26-34.

(8.) Baker LC, London B, Choi BR, Koren G, Salama G. Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res 2000; 86: 396-407.

(9.) Pashmforoush M, Lu JT, Chen H, St. Amand T, Kondo R, Pradervand S, et al. Nkx2-5 pathways and congenital heart disease: loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell 2004;117: 373-86.

(10.) Homburger F. Myopathy of hamster dystrophy: history and morphologic aspects. Ann NY Acad Sci 1979; 317: 1-7.

(11.) Roshchevsky MP, Chudorodova SL, Shmakov ON, Roshchevskaya IM. The cardioelectric field on the pig body surface during initial atrial activity. Doklady Biol Sci 2005; 402: 561-2.

Yu-Jun Lai (1,2,4), Ya-Yu Chen (2), Chio-Pei Cheng (2), Jim Jung-Ching Lin (5), Svetlana L Chudorodova (6), Irina M Roshchevskaya (6), Mikhail P Roshchevskys (6), Yao-Chang Chen (3) Cheng-I Lin (2)

Institutes of (1) Life Sciences & (2) Physiology and (3) Department of Biomedical Engineering, National Defense Medical Center, Taipei (4) Mackay Memorial Hospital, Taiwan, R.O.C. (5) Department of Biological Sciences, University of Iowa, Iowa City, Iowa, U.S.A. (6) Komi Science Center, Ural Division, Russian Academy of Science, Syktyvkar, Russia

Address for Correspondence: Prof. Cheng-I Lin, Ph.D., Institute of Physiology, National Defense Medical Center, Room 6322, no. 161, Min-Chuan Rd., Sec. 6, Neihu, 114, Taipei, Taiwan E-mail: bme03@@ndmctsgh.edu.tw
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Article Details
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Title Annotation:Original Investigation
Author:Lai, Yu-Jun; Chen, Ya-Yu; Cheng, Chio-Pei; Lin, Jim Jung-Ching; Chudorodova, Svetlana L.; Roshchevsk
Publication:The Anatolian Journal of Cardiology (Anadolu Kardiyoloji Dergisi)
Article Type:Clinical report
Geographic Code:4EXRU
Date:Jul 1, 2007
Words:1964
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