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Cardiovascular Research 2006 72(3):412-421; doi:10.1016/j.cardiores.2006.09.010
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Copyright © 2006, European Society of Cardiology

Heavy ion radiation up-regulates Cx43 and ameliorates arrhythmogenic substrates in hearts after myocardial infarction

Mari Aminoa,f,1, Koichiro Yoshiokaa,1, Teruhisa Tanabea, Etsuro Tanakab, Hidezo Moric, Yoshiya Furusawad, Wojciech Zarebae, Masatoshi Yamazakif, Harumichi Nakagawaf, Haruo Honjof, Kenji Yasuif, Kaichiro Kamiyaf and Itsuo Kodamaf,*

aDepartment of Cardiology, Tokai University School of Medicine, Isehara, Japan
bDepartment of Nutritional Sciences, Tokyo University of Agriculture, Tokyo, Japan
cDepartment of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan
dNational Institute of Radiological Sciences, Chiba, Japan
eCardiology Unit, University of Rochester, Rochester, USA
fResearch Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

* Corresponding author. Tel.: +81 52 789 3871; fax: +81 52 789 3890. Email address: ikodama{at}riem.nagoya-u.ac.jp

Received 4 June 2006; revised 12 September 2006; accepted 15 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Objective: Radiation has been shown to enhance intercellular communication in the skin and lungs through an increase of connexin43 (Cx43) expression. If analogous Cx43 up-regulation is induced in the diseased heart, it would provide a new perspective in radiation therapy for arrhythmias. The aim of the present study is to test this hypothesis.

Methods: Non-transmural myocardial infarction (MI) was created in 24 rabbits by microsphere injection into the coronary arteries. Twenty-four rabbits without MI were used as controls. Targeted external heavy ion beam irradiation (THIR; 15 Gy) was applied 2 weeks after MI with an accelerator (HIMAC, Chiba, Japan).

Results: The THIR was associated with an increase of Cx43 mRNA and protein levels in the left ventricle in control as well as in MI rabbits. THIR also increased lateralization of Cx43, which was no longer colocalized with cadherins. In MI hearts, immunoreactive Cx43 signals were reduced in the peri-infarct zone, and the reduction was reversed by THIR. In-vivo epicardial potential mapping on the free wall (64 unipolar electrodes to cover 7x7 mm) in MI hearts revealed reduced conduction velocity, whereas dispersion of the activation-recovery interval (ARI) was increased compared with controls, and these changes were reversed by THIR. The vulnerability for ventricular tachyarrhythmias (VT/VF), which was estimated by programmed stimulation, was increased in MI hearts, and this increased vulnerability to arrhythmias was reversed by THIR.

Conclusions: THIR increases Cx43 expression, improves the conductivity, decreases the spatial heterogeneity of repolarization, and reduces the vulnerability of rabbit hearts to ventricular arrhythmias after MI. THIR could have an antiarrhythmic potential through an improvement of electrical coupling.

KEYWORDS Gap junctions; Connexin43; Heavy ion radiaton; Myocardial infarction; Ventricular arrhythmias; Arrhythmia (mechanisms); Epicardial mapping


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Modalities currently available for treatment and prevention of life-threatening ventricular tachyarrhythmias (VT/VF) are antiarrhythmic drugs, catheter ablation and implantable cardioverter/defibrillator (ICD). The usefulness of these therapeutic options is limited by either low efficiency, intolerable side effects, or impairment of the quality of life (QOL) of the recipient. Fundamentally innovative antiarrhythmic strategies are, therefore, a matter of great concern to cardiologists.

In the heart, gap junctions (GJs) provide the pathways of intercellular current flow, enabling coordinated action potential propagation and contraction. GJ-channels are constructed from connexins (Cx), a multigene family of conserved proteins. In the mammalian heart, connexin43 (Cx43) is the most abundant and ubiquitous. Deranged expression and organization of Cx43 GJs in the ventricular muscles have been demonstrated in a variety of diseased hearts including ischemia, hypertrophy and inflammatory cardiomyopathy [1]. Such GJs remodeling is supposed to create arrhythmogenic substrates by modulating the propagation of excitation. X-ray irradiation has been shown to increase intercellular communication in the mouse skin [2] and rat alveolar epithelial cells in the lung [3] through an increase of expression of Cx43. If analogous up-regulation of Cx43 by radiation is induced efficiently in the diseased heart, it would provide a new perspective in the treatment of arrhythmias.

Radiotherapy using heavy-ions to treat deep-seated cancer was started at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan in 1994 [4]. Charged-ion beams such as accelerated carbon-ions show a unique depth-dose distribution referred to as the Bragg peak in the target matter [5]. Those energetic ion beams decrease in kinetic energy thus reducing the velocity, finally stopping at a defined depth in the target object with high linear energy transfer (LET) [6], indicating a high relative biological effectiveness to X-rays (RBE). This gives advantages to heavy-ions over other radionucleid species in cancer therapy [5,6]. Based on this oncological experience, we hypothesized that such targeted heavy-ion beam irradiation (THIR) could also offer an advantage in causing up-regulation of Cx43 in the ischemic myocardium with minimal damage to the surrounding tissues.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Animal-handling followed the Guide for the Care and Use of Laboratory Animals (NIH Publication 85–23, revised 1996) with procedures approved by the Animal-Experimentation Ethics Committee of the Tokai University.

2.1. Animal model and heavy ion radiation
New Zealand white rabbits (n=48) weighing 3.5–4.0 kg were used. Non-transmural patchy myocardial infarction (MI) was created in 24 rabbits by microsphere injection (15 µm in diameter, 5x105/mL, 3 mL) into the coronary arteries by the transcatheter approach introduced from the carotid arteries [7]. The remaining 24 rabbits served as controls. Two weeks later, each 12 of MI and control rabbits received targeted THIR. Therefore, 4 animal groups were prepared: Control (C), C+THIR, MI, and MI+THIR.

We used carbon-ion beams provided by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at NIRS, Japan [4]. The antero-lateral left ventricular (LV) free wall was focused for irradiation with carbon-ion beams (15 Gy) through the left anterior breast. The dose setting was confirmed appropriate in a pilot study. More details regarding the THIR procedures are described in the online data supplement.

2.2. Histology and immunohistochemistry
Ventricular tissue sections (12 µm thick slices) were fixed and embedded in paraffin. To recognize the MI and fibrosis region, the sections were stained by both hematoxylin/eosin (HE) and azan. The amounts of fibrosis were estimated from the binary images of azan-staining (Fig. 1). For immunostaining, the sections were incubated with an anti-Cx43 mouse monoclonal antibody (Chemicon), and then were treated with the secondary antibody (Alexa Flour 488 conjugated anti-mouse IgG). Some sections were also labeled with Alexa Flour 594 conjugated anti-cadherin, mouse monoclonal antibody (abcam). The immunolabelled sections were examined with a laser confocal microscope (LSM510, Version 2.02), and analyzed with the CLSM macro program (Carl Zeiss).


Figure 1
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  		Fig. 1 Creation of non-transmural patchy myocardial infarction (MI) in rabbit hearts. A, a complete cross section of a low (left top), a moderate (right top) and a high magnification (bottom) of the ventricles 4 weeks after the creation of MI stained with HE/azan. Patchy fibrotic tissues (blue) are widely distributed in the subendocardial (a), mid-myocardial (b) and subepicardial (c) layers of the left ventricle (LV). B, 3-dimensional illustration of fibrotic tissue distribution (blue) in the same heart.

 
2.3. Real-time PCR and Western blotting
To quantify mRNA expression of Cx43 in the LV free wall, we performed a real-time PCR assay (Perkin-Elmer ABI Prism7700) [8]. GAPDH mRNA was used as an internal control. Sequence of PCR primers and sequence-specific probes are shown in the online data supplement.

The amount of Cx43 protein was evaluated by Western blotting [9]. The intensity of the Cx43 bands was quantified by densitometry and normalized to {alpha}-tubulin as the control.

2.4. In-vivo experiments
In-vivo electrophysiology experiments were conducted following open thorax surgery under anesthesia ({alpha}-chloralose, 80 mg/kg and urethane, 1000 mg/kg) [10]. An array of 8x8 monopolar electrodes (64Map) to cover 7x7 mm square (interpolar distance, 1 mm) was put on the LV wall between the left anterior descending branch (LAD) and left circumflex branch (LCx) of coronary arteries (Fig. 5A) [10]. The 64Map signals recorded simultaneously were acquired and processed by a computer. Activation time (AT) was defined as the interval from the beginning of QRS to the initial sharp negative deflection (min dV/dt). Recovery time (RT) was defined as the interval from the beginning of QRS to max dV/dt of T wave. The time difference between AT and RT in each electrogram was measured for the activation-recovery interval (ARI) as an index of action potential duration [11]. The dispersion of ARI (ARID) in the mapped area was estimated by the standard deviation of ARI at 64 electrodes [12]. The earliest AT within the electrode array was set down as 0 ms. Total activation time (TAT) was assessed as the delay between activation of the first and activation of the last electrode [12].


Figure 5
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  		Fig. 5 Epicardial LV potential mapping. A, Sixty four monopolar electrodes were arranged as an 8x8 array to acquire the signals. Activation time (AT) at the recording site was identified by the initial sharp negative deflection (min dV/dt) of QRS. Recovery time (RT) was identified by the maximum upstroke slope (max dV/dt) of T wave. The time difference between AT and RT in each electrogram was measured for activation-recovery interval (ARI). B, Representative maps of AT and ARI during sinus rhythm. Total activation time (TAT) was measured from the maximal difference of AT. The dispersion of ARI (ARID) was estimated by the standard deviation of ARI at 64 electrodes. C, Anisotropic conduction properties were examined under constant pacing from the top middle of the electrode array. A line for longitudinal (L) propagation was drawn from the pacing site to the outer edge of the map, so as to cross the most widely spaced isochrones. A second line for transverse (T) propagation was drawn perpendicular to the first one. D, Representative maps of AT during the constant pacing. Conduction velocity in L and T direction (CVL, CVT) were measured from the isochrone maps.

 
In order to assess the anisotropic conduction property, LV was paced at a cycle length of 200 ms (pulses of 1 ms duration and 1.2 times the threshold) via a pair of contiguous bipolar electrodes placed in the middle on the upper edge of the electrode array. Longitudinal (L) and transverse (T) directions of propagation were determined from the isochrone maps [9]. Conduction velocity (CV) was determined by linear regression of the isochrone distance vs. AT.

Propensity to VT/VF was tested by programmed electrical stimulation under norepinephrine infusion (0.1 µg/kg/min, i.v.). A pair of bipolar electrodes was placed on the epicardial surface near the LV apex. Following 5 basic stimuli (S1) at a cycle length of 200 ms, triple extra-stimuli (S2–S4) of double threshold were applied at progressively shorter coupling intervals.

2.5. Data analysis
Data are presented as means±SD. Data sets containing multiple groups were analyzed by 2-way layout analysis of variance and Bonferroni type multiple comparisons. Differences were considered statistically significant at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
3.1. Standard light microscopy
Pathological features of rabbit hearts 4 weeks after creation of non-transmural MI by the microsphere injection method were analyzed in sections stained with HE and azan. Representative results (without radiation) are shown in Fig. 1. Multiple, small patchy infarctions with fibrosis surrounded by noninfarcted myocardium were recognized throughout the whole LV, and in part in the right ventricular (RV) free wall. Although the entire thickness of LV wall was involve, the fibrosis was more marked in the subendocardial region compared with subepicardial region. Qualitatively similar results were obtained from the hearts with and without THIR. The fibrosis areas estimated from the azan-staining of 4 whole cross sections in each heart from the base to the apex (Fig. 1) were 12.7±4.4% and 12.2±4.8% in MI and MI+THIR groups, respectively (n=5, P>0.05). Thus, THIR did not affect the amounts of fibrosis.

3.2. Immunostained Cx43 GJs in LV myocardium
Confocal microscopy for Cx43 immunolabeling was carried out in 6 hearts from all the 4 animal groups. Fig. 2A shows the representative effects of THIR in the control heart (without MI creation). Longitudinal sections of LV free wall myocardium were labeled for Cx43 (stained green) and cadherin (stained red). In Control, Cx43 formed clusters of punctate immunofluorescence domains confined to well-organized intercalated disks running across the longitudinal axis (panel a, arrows). Cadherin labeling showed a similar distribution to Cx43 (panel b). Double staining revealed the co-existence of Cx43 GJs and cadherin-containing fascia adherents confined to the intercalated disks, giving rise to their yellowish staining (panel c). In a rabbit 2 weeks after radiation (C+THIR), in contrast, Cx43 labeling was distributed not only at the intercalated disk but also at the lateral aspect of myocytes (panel d, arrowheads), whereas the normal distribution of cadherin was preserved (panel e). Double staining confirmed the dissociation of Cx43 from the intercalated disks (panel f). Similar derangements (lateralization) of Cx43 distribution were recognized in all C+THIR group. Fig. 2B shows the proportion of total cell area occupied by Cx43 immunoreactive signals. The radiation resulted in a significant increase in the Cx43 immunoreactive signals of 76%. Fig. 2C shows the proportion of Cx43 labeling at the lateral cell surface over the total labeling. The value in C+THIR was significantly higher (by 535%) than Control.


Figure 2
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  		Fig. 2 Immunostained Cx43 gap junctions and fasciae adherentes in the LV myocardium of rabbits without MI. A, An LV myocardium sectioned longitudinally was labeled for Cx43 (green) and cadherin (red). Top, a section from a control heart; bottom, a section from a heart after targeted heavy ion irradiation (THIR). All images are single optical slices (confocal laser microscopy). Scale bar, 20 µm. B, The proportion of the total cell area occupied by Cx43 immunoreactive signal. Average values of 5 fields in each sample was obtained, and then means±SD of the values were calculated for each 6 samples. *P<0.05 vs. Control. C, Changes in the proportion of Cx43 labeled at the intercalated disk region (ID) and lateral cell surface (LS). (means±SD). Data were obtained from 12 rabbits (6 in each group). *P<0.05 vs. Control.

 
Fig. 3 show the effects of THIR on MI hearts. Representative changes of Cx43 immunolabeling patterns were compared in Fig. 3A at a low and a high magnification. In an MI heart (left), normal Cx43 labeling confined to the intercalated disks was preserved in the area distant from the infarcted tissue (patchy loss of myocytes indicated by arrowheads) (panels a, and c). In the area neighboring the infarcted tissue, Cx43 labeling was characterized either by prominent disorganization (panel e) or depletion (panel d). In an MI+THIR heart (right), immunoreactive Cx43 signals were increased throughout all of the LV tissues. This up-regulation was recognized not only in the normal zone distant from the patchy infarcted tissue (panel f), but also in the peri-infarct regions (panels g and h). The punctate Cx43 immunolabeling was distributed over the intercalated disk regions as well as lateral abutments of myocytes. Fig. 3B shows the proportion of total cell area occupied by the Cx43 immunoreactive signal obtained from the MI and MI+THIR groups. The irradiation resulted in a significant increase in Cx43 immunoreactive signals of 76%.


Figure 3
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  		Fig. 3 Immunostained Cx43 gap junctions in the LV myocardium of rabbits with non-transmural MI. A, Left, a section from an MI heart; right, a section from a heart from MI+THIR. All images are single optical slices. Scale bar is 100 µm in panels (a) and (b), and 20 µm in panels (c) through (h). B, The proportion of total cell area occupied by Cx43 immunoreactive signal. Average values of 5 fields in each sample was obtained, and then means±SD of the values were calculated for each 6 samples. Data were obtained from 12 rabbits (6 in each group). #P<0.05 vs. MI.

 
3.3. Expression of Cx43 mRNA and protein in the LV myocardium
The Cx43 mRNA level by a real-time PCR assay in the C+THIR specimens was significantly larger than that in Control specimens (by 18%) (Fig. 4A). The level in MIs, in contrast, decreased significantly compared with Controls (by 33%). Irradiation of MI rabbits (MI+THIR) reversed the down-regulation of Cx43 mRNA and increased by 88% compared with MI.


Figure 4
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  		Fig. 4 Cx43 mRNA and protein levels. A, Cx43 mRNA levels were estimated by a real-time PCR and normalized to GAPDH. Top: Representative Cx43 PCR amplification plots for reactions. Change in fluorescence signal ({delta}Rn) is plotted against cycle number. Bottom: Cx43 mRNA levels normalized to GAPDH mRNA. Values are means±SD of each 5 rabbits in the 4 animal groups. B, Cx43 protein levels were estimated by Western blotting and normalized to {alpha}-tubulin. Values are means±SD of each 5 rabbits in the 4 animal groups. *P<0.05 vs. Control. #P<0.05 vs. MI.

 
Cx43 protein amounts were estimated by Western blotting (Fig. 4B). The Cx43 antibodies recognized two bands migrating between 42 and 47 kDa. The Cx43 immunoblot signals were quantified by densitometry, and the intensity was normalized to that of {alpha}-tubulin on the same membrane. Irradiation of control rabbits (C+THIR) caused a significant increase in the total amount Cx43 protein by 88% compared with Control animals. In MI rabbits, the total Cx43 protein levels significantly decreased compared with Controls (by 95%), and irradiation of MI rabbits (MI+THIR group) increased the level by 133% in comparison with unirradiated MI rabbits. We also estimated the density of the higher and lower molecular weight bands separately, which correspond to phosphorylated (P) and non-phosphorylated (NP) isoforms of Cx43, respectively. Irradiation caused appreciable increases in both Cx43-P and Cx43-NP in Control as well as in MI rabbits. There were no significant differences among the four groups in the averaged ratio of P/NP (Control, 0.60; C+THIR, 0.84; MI, 1.00; MI+THIR, 0.96). Taken together, heavy-ion irradiation caused significant up-regulation of both mRNA and total protein levels of Cx43 in control as well as in MI hearts.

3.4. In-vivo electrophysiology
Fig. 5B illustrates representative AT and ARI maps on the LV anterior surface during the sinus rhythm (heart rate 197–238 bpm). In a Control rabbit, the activation proceeded quickly from paraseptal to lateral direction; TAT in the mapped area was 12 ms. ARIs were almost uniform, giving rise to a small ARID of 17 ms. In a C+THIR rabbit, the activation proceeded similarly (TAT 10 ms), but ARIs were homogeneously prolonged with ARID of 18 ms. In an MI rabbit, the activation proceeded much more slowly (TAT 32 ms). The ARI map showed tremendous regional variation, and ARID was increased up to 32 ms. In an MI+THIR rabbit, the AT map showed faster propagation (TAT 14 ms), and ARIs were more homogeneous than MI, causing less ARID (21 ms).

Data obtained from the 4 groups are summarized in Table 1. TAT was unchanged in C+THIR, but increased significantly in MI (by 89% from Control). The TAT in MI+THIR was significantly less than MI (by 42%), suggesting a reversal of the conduction delay. Both RI and ARI were significantly increased in C+THIR (by 10% and 11%, respectively). MI caused an appreciable prolongation of RT without affecting ARI compared with Control. In MI rabbits, RT and ARI were increased moderately after irradiation (MI+THIR), but the differences did not reach a statistical significance. In control rabbits, ARID was unaffected by irradiation. MI caused a significant increase in ARID (by 81%), and irradiation reversed the change.


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  		Table 1 In-vivo electrophysiology

 
Anisotropic conduction properties in the epicardial surface were examined in the 4 animal groups under constant pacing (cycle length of 200 ms) (Fig. 5C, D). In Control, the activation front proceeded at the highest speed in a direction parallel (longitudinal, L) to the subepicardial fiber orientation and at the slowest speed in a direction perpendicular to that (transverse, T). The isochrones showed an elliptical activation pattern. In the heart from a C+THIR rabbit, the elliptical pattern was less marked (more circular) because of acceleration of T propagation. In the heart from an MI rabbit, both L and T propagations were slowed down compared to Control, and the elliptical shape was slightly enhanced. In an MI+THIR rabbit, the activation pattern was more circular compared with MI alone, because of an acceleration of T propagation, which was more remarkable than the acceleration of L propagation. CVs in L and T directions (CVL, CVT) are summarized in Table 1. In control rabbits, irradiation caused a significant increase only in CVT (by 49%), resulting in a significant decrease of the anisotropic ratio (L/T). In MI rabbits, both CVL and CVT were decreased compared with Control. Irradiation to MI rabbits (MI+THIR) caused significant increases of both CVL and CVT, but the change in CVT (by 50%) was larger than that in CVL (by 27%), resulting in a moderate reduction of L/T.

VT/VF induction was attempted by programmed stimulation in each 5 rabbits from the 4 animal groups. Representative ECG records in a Control, an MI and an MI+THIR rabbit are shown in Fig. 6A. Fig. 6B shows AT maps of 2 sequential beats during the VT/VF documented from the MI and MI+THIR rabbits. Fig. 6C summarizes the VT/VF incidence. In Control and C+THIR, no VT/VF was induced. In MI, 2 VF and 2 VT (one sustained >30 s, the other non-sustained) were induced. In MI+THIR, only one non-sustained VT (NSVT) was induced. Activation patterns during the NSVT were much more homogeneous than that during VF documented in MI rabbits (Fig. 6B).


Figure 6
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  		Fig. 6 Induction of VT/VF by programmed stimulation under norepinephrine infusion (0.1 µg/kg/min, i.v.). A, Representative ECG recordings in Control, MI and MI+THIR rabbits. Following 5 basic stimuli (S1) at a cycle length of 200 ms, triple extra stimuli (S2–S4) were applied at progressively shorter coupling intervals. B, AT maps of two sequential beats during the VF and VT episodes in the MI and MI+THIR rabbits as presented in A. C, Incidence of VT/VF elicited by the programmed stimulation. SVT: sustained VT (>30 s), NSVT: non-sustained VT (<30 s). VFs were all sustained.

 
3.5. Safety of irradiation
In both control and MI rabbits, THIR did not affect the hemodynamic parameters in echocardiography 2 weeks after the irradiation. The LV function in MI hearts was inhibited significantly compared with Control, and the irradiation did not ameliorate the hemodynamic parameters (online data supplement). Blood cell counts and chemistry were unaffected by the irradiation. However a circumscribed loss of hair began to appear on the left anterior chest soon after irradiation and remained for a year.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
4.1. The novel findings
Immunoreactive Cx43 fluorescent signals of MI hearts were reduced and disorganized notably. In addition, the Cx43 mRNA level and the amount of Cx43 protein in LV tissue, which were estimated by a real-time PCR and Western blotting, respectively, were markedly reduced in MI hearts. Most of these changes were reversed 2 weeks after THIR. In-vivo 64Map revealed that a reduction of CV in MI hearts was reversed by THIR. An increase of ARID in MI hearts was reversed by THIR. An increase of vulnerability for VT/VF induction by programmed stimulation in MI hearts was also reversed by THIR. All of these observations can be interpreted most likely by an improvement of electrical coupling of cardiac myocytes, causing a smoother propagation of excitation and homogeneous repolarization in the ventricle after MI.

Achieving up-regulation of Cx43 in the myocardium might be a target for novel antiarrhythmic therapies. The use of radiation to up-regulate the decreased Cx43 in diseased hearts has never been considered. Radiotherapy might be one of the only potential approaches for future therapeutic strategy through achieving an increase in the electrical coupling of cardiac cells via the up-regulation of GJs.

4.2. Functional consequences of Cx43 up-regulation and lateralization
In normal as well as in MI hearts, THIR caused up-regulation of Cx43 and an increase of Cx43 distribution at the lateral cell abutments (lateralization). These changes would have a variety of functional consequences. The activation maps under constant pacing showed that the irradiation increased CVs and the effect was greater in T than L propagation, giving rise to a reduction of the anisotropic ratio (L/T). This observation suggests that Cx43 up-regulated and distributed more laterally may form functional GJs even though Cx43 was dissociated from cadherin. In rat atria in which atrial fibrillation (AF) was induced by 24 h of rapid pacing, Polontchouk et al. [13] showed up-regulation and marked lateralization of immunoreactive Cx43, like in human AF patients. The Cx43 remodeling was associated with a greater increase of CVT than CVL, resulting in a reduction of the anisotropic ratio. In experiments using isolated normal ventricular myocardium, pharmacological uncoupling of GJs (by heptanol or palmitoleic acid) was shown to cause greater inhibition of T propagation than L propagation [14,15]. A higher susceptibility of T propagation than L propagation in response to GJ uncoupling was also demonstrated in a computer simulation [16]. Accordingly, the total up-regulation of Cx43 by THIR could also contribute to the preferential improvement of T propagation.

It appears that lateralization of gap junctions is a prominent feature of diseased hearts, including a variety of ischemic and hypertrophic cardiomyopathies [1,16]. Functional implication of this feature is variable. In experiments using aged rabbits, Dhein and Hammerrath [17] found reduced CVT and enhanced anisotropy (CVL was preserved). Those changes were associated with prominent increase of fibrosis separating myocardial cells and lateralization of Cx43. The extensive fibrosis might have offset the effects of GJ lateralization. Alternatively, the displaced Cx43 GJs could be degraded and non-functional [1]. Since we used young rabbits in the present study, the control hearts with and without THIR had no appreciable collagen strands separating myocardial fibers. In MI hearts, in contrast, there existed substantial amounts of fibrosis at the regions of patchy infarction. Despite of such fibrosis, THIR improved the T propagation.

As to the role enhanced GJ lateralization in arrhythmogenesis, there are 2 possibilities. If the GJs function normally, they would ameliorate the inhibition of T propagation resulting from functional and structural uncoupling of cardiac cells, and may reduce the risk for microscopic anisotropic reentry based on the extremely slow T propagation [18,19] and the lateral inhomogeneity of repolarization. The present results seem consistent with this prediction. If the lateral GJs are degraded and non-functional, the conductivity and electrical homogeneity of the heart will be hampered by the dislocation in favor of reentrant excitations. In a simulation study using a two-dimensional model of myocardial cell architecture, Spach and Heidlage [20] suggested the stochastic nature of normal propagation at a microscopic level based on the normal polar distribution of GJs provides a considerable protective effect against arrhythmias by reestablishing the general trend of wave-front movement after small variations in excitation events occur.

Cx43 is a phosphoprotein, and phophorylation/dephosphorylation of Cx43 plays important roles in the regulation of Cx43 protein turnover dynamics (trafficking, plaque assembly, disassembly and degradation) as well as GJ channel gating properties [21]. We estimated the relative expression of P and NP isoforms of Cx43 from 2 bands recognized in Western blotting. The results may suggest that THIR increases both Cx43-P and Cx43-NP without affecting their P/NP ratio. However, more extensive Western blots using isoform-specific Cx43 antibodies will be required to resolve the issue.

4.3. Mechanisms of Cx43 up-regulation by irradiation
In the field of oncology, it has been demonstrated in many in-vitro and in-vivo studies that intercellular communication is enhanced by photon [2,3] or ionizing radiation [22,23] via up-regulation of Cx43 GJs at mRNA and protein levels. The increase of intercellular communication is believed to play an important role for the enhancement of radiation-induced effects such as modulation of gene expression, mutagenesis and cell survival (bystander effect) [24–26]. As to the molecular mechanisms responsible for the radiation-induced Cx43 up-regulation, the information available is still limited. Using deletion and site-directed mutagenesis analyses in human fibroblasts and HeLa cells, Glover et al. have demonstrated that 2 consensus sites, nuclear factor of activated T-cells (NFAT) and activator protein (AP1), are responsible for the major activation of the Cx43 promoter in response to low doses of ionizing radiation [23] A similar molecular mechanism could be involved in the THIR-induced up-regulation in mammalian cardiac cells, but the issue remains to be investigated.

4.4. What other methods could be considered to achieve similar results?
Several procedures have been proposed by previous investigators to increase the electrical coupling of ventricular myocytes through an increase of Cx43 protein. Those include endothelin-1, angiotensin-II, thyroid hormones, AAP10 and nitrofen [27–30]. As to the therapeutic challenge to ameliorate arrhythmogenic substrates, however, all of these procedures have significant limitations in their clinical feasibility because of their low efficiency, undesirable cardiovascular and other side effects. We believe that THIR might be the first clinically feasible procedure, although further experimental studies using large animals will be required to substantiate this proposal.

4.5. Limitations
1) Because the rabbit heart is smaller and the heart rate is faster than human, higher irradiation accuracy was demanded in the present study. It was practically difficult to synchronize the heart motion completely to correspond to the radiation release. In addition to the targeted anterolateral LV free wall, other regions of LV might have been subjected to the irradiation. 2) The time dependent expression of Cx43 and the minimum irradiation energy for the appearance of the first antiarrhythmic effect were not examined.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
Appendix A. Supplementary data
 Acknowledgments
 References
 
Heavy-ion energy increases Cx43 expression of the ventricle in MI rabbits and consequently improves the conductivity, decreases the spatial heterogeneity of repolarization, and reduces the VT/VF vulnerability. Targeted heavy-ion irradiation to the heart could have the potential to become a new antiarrhythmic preventive therapy for MI patients through the restoration of cell-to-cell electrical coupling.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
Acknowledgments
 References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.09.010.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
References
 
This work was supported by Grants-in-Aid for Scientific Research (B) 17390236 from JSPS and Tokai University School of Medicine Research Aid. We are grateful to many colleagues for their technical support: Atsushi Matsuzaki, Kazutane Usui, Yoshiaki Deguchi, Yuji Ikari, Nobue Kumaki, Sachie Tanaka, Noboru Kawabe, Hideaki Hasegawa, Yoshiro Shinozaki, Jobu Itoho in Tokai University; Jong-Kook Lee, Yoshiko Takagishi, Akiko Matsumiya, Kyoko Harada, Mayumi Hojo in Nagoya University; Takeshi Murakami, Kumie Nojima in the HIMAC Cooperative Research Project; Norio Sugimoto in the SANWAKAGAKU laboratory; Norihiko Mishima, Masaya Sakai, Satoshi Yamazaki in FUKUDA DENSHI. Co Ltd; Daisuke Araki, Takashi Akamatsu, Michinari Kaneko in UNIQUE MEDICAL. Co Ltd; and Daisuke Nakata in Ela Medical. Co Ltd.


   Notes
 
1 The first two authors contributed equally to this work. Back

Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Appendix A. Supplementary data
 Acknowledgments
 References
 

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