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Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-

Cardiovascular Institute¹ and Department of Cell Biology and Physiology,² University of Pittsburgh, Pittsburgh, Pennsylvania; and Department of Medicine,³ Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 21 July 2004 ; accepted in final form 16 May 2005

	ABSTRACT

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Transgenic mice overexpressing the inflammatory cytokine TNF- in the heart develop a progressive heart failure syndrome characterized by biventricular dilatation, decreased ejection fraction, decreased survival compared with non-transgenic littermates, and earlier pathology in males. TNF- mice (TNF1.6) develop atrial arrhythmias on ambulatory telemetry monitoring that worsen with age and are more severe in males. We performed in vivo electrophysiological testing in transgenic and control mice, ex vivo optical mapping of voltage in the atria of isolated perfused TNF1.6 hearts, and in vitro studies on isolated atrial muscle and cells to study the mechanisms that lead to the spontaneous arrhythmias. Programmed stimulation induces atrial arrhythmias (n = 8/32) in TNF1.6 but not in control mice (n = 0/37), with a higher inducibility in males. In the isolated perfused hearts, programmed stimulation with single extra beats elicits reentrant atrial arrhythmias (n = 6/6) in TNF1.6 but not control hearts due to slow heterogeneous conduction of the premature beats. Lowering extracellular Ca²⁺ normalizes conduction and prevents the arrhythmias. Atrial muscle and cells from TNF1.6 compared with control mice exhibit increased collagen deposition, decreased contractile function, and abnormal systolic and diastolic Ca²⁺ handling. Thus abnormalities in action potential propagation and Ca²⁺ handling contribute to the initiation of atrial arrhythmias in this mouse model of heart failure.

heart failure; atrium; atrial fibrillation; cytokines

We have previously used optical mapping of voltage and calcium in ventricles from the TNF1.6 mice to show prolongation of action potential duration (APD), prolongation of calcium transient duration, and elevated diastolic and depressed systolic calcium (33). Premature beats had depressed action potential amplitude and slowed conduction velocities that contributed to initiation of reentrant arrhythmias. Lowering extracellular calcium reversed the abnormalities and prevented arrhythmia induction. We now show that these mice develop spontaneous atrial arrhythmias on ambulatory telemetry monitoring and have inducible atrial arrhythmias during in vivo and ex vivo electrophysiological testing. They also develop increased atrial collagen deposition, contractile dysfunction, and abnormal systolic and diastolic Ca²⁺ handling. These abnormalities taken together may contribute to the initiation and maintenance of reentrant atrial arrhythmias in heart failure.

	MATERIALS AND METHODS

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Breeding of TNF1.6 transgenic mice. All studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Heterozygous TNF1.6 transgenic mice were bred with FVB controls to generate TNF1.6 mice and wild-type littermate controls. Mice aged 2 to 9 mo were used for the current studies.

Morphology and immunohistochemistry. Hearts used for immunohistochemistry and functional studies (isolated atria or single atrial myocytes) were isolated from mice deeply anesthetized with isoflurane, heparinized (50 units ip), and then euthanized by cervical dislocation. Hearts from 2- to 9-mo-old transgenic and wild-type female mice were fixed in ice-cold 2% paraformaldehyde, immersed in ice-cold 30% sucrose, and flash-frozen in OCT (Miles) medium with isopentane cooled by liquid nitrogen. Four-chamber sections of the hearts were stained with hematoxylin and eosin. Collagen immunohistochemistry was performed with rabbit antibodies against type I collagen (Chemicon, Temecula, CA) and appropriate secondary antibodies. Sections were costained with phalloidin Alexa Fluor 488 (Molecular Probes, Eugene, OR) and analyzed using a fluorescence microscope. The collagen percent fractional area was calculated as the visual field area occupied by collagens divided by the sum of the area stained by collagen and phalloidin (48).

Measurement of contractile function of isolated atria. Hearts isolated from 6- to 9-mo-old TNF1.6 and control female mice were placed in a Krebs-Henseleit solution containing (in mM) 141 NaCl, 25 NaHCO₃, 5 HEPES, 1.2 NaH₂PO₄, 1.0 MgSO₄, 5.4 KCl, 50 dextrose, 2.0 CaCl₂, and 30 2,3-butanedione monoxime, bubbled with 95% O₂ and 5% CO₂ (pH 7.4). Under a dissecting microscope, left atria were isolated from the heart. With the use of fine sutures, a small loop was formed and attached to the connective tissue at the valvular end of the atrium and the other end was clamped with a minivessel clamp. Atria were then transferred to a temperature-controlled (36°C) experimental chamber perfused with the Krebs-Henseleit solution (without 2,3-butanedione monoxime) and attached to a force transducer (model 405A, Aurora Scientific, Ontario, Canada) on one side and to a micromanipulator on the other side. During a 60-min equilibration period, the atrium was electrically stimulated at 0.5 Hz via platinum electrodes and gradually stretched until maximal contractility was reached. Thereafter, the muscle length was adjusted to produce 90% maximal force, and the interval-force relationship was examined at frequencies between 1 and 6 Hz. Data were acquired using an IonOptix setup and analyzed with IonWizard 5.0 software (IonOptix, Milton, MA). Cross-sectional area (CSA) of the atrium was determined from the length and weight of the muscle after the experiment, assuming a density of 1.063 (26).

Ambulatory recording of arrhythmias in mice. Radiotelemetry electrocardiographic monitors (Data Sciences) were implanted subcutaneously on the backs of TNF1.6 and control mice using Avertin anesthesia as previously described (33). The mice were allowed to recover for at least 6 days, at which point 24 h of telemetry were recorded at 400 Hz and saved on disk. Monitoring was then continued for up to 4 mo. All telemetry data were scanned for atrial arrhythmias by hand or by using a computer-based arrhythmia detection system. At least 95% of the data for each mouse was adequate for analysis.

In vivo cardiac electrophysiological testing. A total of 69 mice were studied. Young (age = 15 ± 2 wk; male: 6 TNF1.6, 15 control; female: 12 TNF1.6, 10 control) and old (age = 27 ± 4 wk; female: 14 TNF1.6, 12 control) mice underwent blinded in vivo closed-chest, electrophysiological testing before and after -adrenergic stimulation (isoproterenol 2 ng/g ip). Older TNF1.6 male mice were not studied due to high mortality rates before the age of 4 mo.

As previously described, mice were housed in cages in a facility with 12 h-12 h light/dark cycles at a temperature of 24°C, fed rodent chow, and allowed free access to water (38, 39). Mice were anesthetized by intraperitoneal administration of 2.5% Avertin (0.015 ml/g body wt). A six-lead electrocardiogram was obtained by placing 24-gauge needles subcutaneously in each limb. A right external jugular vein cutdown was performed and a 2-Fr octapolar catheter (NuMed, Hopkinton, NY) was advanced to the right ventricular apex until a bipolar His bundle electrogram could be recorded from the middle electrodes. All surgical procedures were done under a surgical microscope (model SMZ 1B, Nikon). After the procedure, the hearts of the mice were excised and weighed.

All intracardiac signals were sampled at 2 kHz and amplified and filtered at 30–500 Hz. Surface signals were filtered at 0.01–100 Hz (Labsystem Duo Bard Electrophysiology, Lowell, MA). Baseline cardiac cycle intervals were measured in all mice, including the cycle length (CL), PR, QRS, QT, AH, and HV intervals. The AH interval was defined as the time from the local atrial deflection to the His deflection on the intracardiac electrograms, and the HV interval was defined as the time from the beginning of the His deflection to the beginning of the QRS deflection on the surface electrogram. Atrial and ventricular pacing thresholds were then determined, and pacing was conducted using 2.0-ms pulse widths at twice diastolic threshold (Bloom stimulator, Fisher Imaging, Reading, PA). Sinus node function was evaluated by measuring sinus node recovery time (SNRT) after the right atrium was paced at a CL of 100 ms for 60 s and correcting it for the baseline heart rate (SNRTc = SNRT – CL). Atrioventricular (AV) and VA Wenckebach and 2:1 CLs were determined. Programmed atrial and ventricular stimulation was performed by delivering a premature stimulus after the eighth stimulus in a drive train. Effective refractory periods as well as functional refractory periods were determined at a CL of 100 ms for both the AV and VA conduction. Ventricular effective refractory period was also determined at a drive CL of 100 ms. Premature atrial and ventricular stimulation curves were constructed and compared between the study groups. Discontinuity or jump was defined as a 25-ms increase in AH or VA for a 5-ms decrease in A₁A₂ or V₁V₂, respectively. Programmed atrial and ventricular stimulation, consisting of burst pacing at CLs of 100 to 50 ms in decrements of 10 ms, and of programmed stimulation with single, double, and triple extrastimuli at a drive CL of 100 ms with a coupling interval greater or equal to 30 ms, was performed in both atria and ventricles. Inducibility was predefined as 10 beats of supraventricular or ventricular tachycardia after the last pacing stimulus. Five to 10 min after the administration of -adrenergic stimulation (isoproterenol 2 ng/g ip), the electrophysiology protocol was repeated. Measurements of all parameters were done by a blinded observer.

Optical mapping of action potentials. Mice were anesthetized with fluothane, heparinized (50 units ip), and then euthanized. The heart was then rapidly excised, cannulated, and placed in a chamber specifically designed to immobilize, pace, and focus an image of the epicardial surface of the heart on a photodiode array as previously described (5, 13, 14). The perfusate contained (in mM) 141 NaCl, 25 NaHCO₃, 5 HEPES, 1.2 NaH₂PO₄, 1.0 MgSO₄, 5.0 KCl, 50 dextrose, and 1.8 CaCl₂ (pH 7.4), bubbled with 95% O₂-5% CO₂. Perfusion pressure was maintained at 60–80 mmHg and the temperature was maintained at 37°C. Hearts were stained with the voltage-sensitive dye pyridinium, 4-[2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)]-hydroxide (di-4-ANEPPS, 10–15 µl of a 3 mM stock solution in DMSO) delivered as a bolus through the port of a bubble trap, which resulted in homogeneous dye loading throughout the heart and action potentials that were stable for up to 4 h (5). Atrial and ventricular contraction were initiated by programmed stimulation from one of the Teflon-coated silver wires brought into contact with the heart on the epicardial surface of the atrium or the ventricle at the apex, midventricle, and base. Pacing was performed at twice diastolic threshold. The electrodes were also used to record electrograms.

Light from a tungsten halogen lamp was collimated, passed through an interference filter (520 ± 20 nm), and focused on the surface of the stained heart. The fluorescence from the dye was passed through a cut-off filter (>630 nm) and focused on the 124-element photodiode array. The array viewed an area of 4 x 4 mm, including the atria and part of the ventricles, meaning that each element recorded electrical activity from a region of 310 x 310 µm with a depth of field of 40 µm. The photocurrent of each photodiode was passed through a current to voltage converter and a second stage amplifier, digitized with temporal resolution >1 kHz, and stored in computer memory. Action potential upstrokes, downstrokes, and conduction velocities were used to generate maps of activation and repolarization using custom software written in Interactive Data Language (5).

Measurement of intracellular Ca²⁺ in isolated atrial myocytes. Single left atrial myocytes were enzymatically isolated from hearts of 3-mo-old male TNF1.6 mice and control littermates or from 6- to 9-mo-old female TNF1.6 and control mice, using a collagenase-based digestion technique described previously for isolation of ventricular myocytes in these mice (21). Intracellular Ca²⁺ (Ca_i²⁺) activity was indexed by fura 2 fluorescence ratio in cells loaded with the acetoxymethyl ester of fura 2, as described previously in ventricular myocytes (21). The experiments were carried out at 23–25°C. The myocytes were bathed in a modified Tyrode solution containing (in mM) 137 NaCl, 15 glucose, 5.4 KCl, 1.3 MgSO₄, 2.0 CaCl₂, and 20 HEPES; pH adjusted to 7.4 with NaOH. Data were acquired and analyzed using an IonOptix (IonOptix, Milton, MA) hardware and software. Fura 2 was excited at the wavelengths of 360 and 380 nm as described previously (Figs. 7–8 and Table 5) (21).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Representative recordings of intracellular free Ca2+ transients indexed by the fura 2 fluorescence ratio (bottom) and their first derivative (top) in single myocytes isolated from left atria of control male (3-mo-old) and female (6- to 9-mo-old) mice and age-matched TNF1.6 mice. Cells were electrically stimulated at 0.5 Hz in presence of 2 mM extracellular Ca2+ at 25°C.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Representative recordings of intracellular free Ca2+ activity indexed by the fura 2 fluorescence ratio (bottom) and its first derivative (top) in single myocytes isolated from left atria of 3-mo-old control (A) and TNF1.6 male (B) mice. Occurrence and configuration of spontaneous Ca2+ transients was examined during 30 s rest after stimulation at 4 Hz.

View larger version (11K): [in this window] [in a new window]   Fig. 9. Sarcoplasmic reticulum Ca2+ content in atrial myocytes. A: representative recordings of action potential- and caffeine (CAF)-induced intracellular Ca2+ transients indexed by the fura 2 fluorescence ratio in single myocytes isolated from the left atria of 6-mo-old female control and TNF1.6 mice. Cells were electrically stimulated at 0.5 Hz in the presence of 2 mM extracellular Ca2+. CAF (20 mM in pipette) was rapidly applied from a micropipette onto the cell in lieu of an electrical stimulus. B: amplitude (systolic-diastolic) of fura 2 fluorescence ratio transients induced by sarcoplasmic reticulum Ca2+ depletions with CAF in experiments illustrated in A. Values are expressed as means ± SE. Analyses were performed on 19 cells from 3 hearts (WT) and on 15 cells from 3 hearts (TG). *P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Atrial morphology and collagen deposition. Cardiac-specific overexpression of TNF- in this model is associated with hypertrophy and dilatation of both the ventricles and atria (Fig. 1). The average weight of isolated left atrium (after surgical removal of an organized blood thrombus, typically observed in TNF1.6 atria) was 3.6-fold larger in 6- to 9-mo-old female TNF1.6 versus control mice (Table 1). Immunohistochemical measurements showed marked collagen deposition in TNF1.6 versus control atria of males (10 wk of age) and females (both 10 and 30 wk of age) (Fig. 2). The percent fractional area found as collagen was 10- to 20-fold higher in TNF1.6 versus control atria (both left and right) in both sexes. Of note, atrial collagen deposition was apparent in hearts from young female TNF1.6 mice before marked ventricular enlargement or hypertrophy. In all cases, no differences were observed in collagen deposition between left and right atria. This type of atrial remodeling (i.e., hypertrophy, dilatation, and fibrosis) has been implicated in both impaired contractile function (48, 23) and arrhythmogenesis (10, 36, 37, 31).

View larger version (55K): [in this window] [in a new window]   Fig. 1. Morphological changes consequent to cardiac-specific overexpression of TNF-. A: representative hearts from 6- to 9-mo-old female transgenic (TNF1.6) and wild-type (WT, control) mice. LA, left atrium. B: 4-chamber sections of hearts stained with hematoxylin and eosin from male and female hearts at different ages.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Fibrotic remodeling of atria overexpressing TNF-. A: immunohistochemical staining of 10-wk-old female right atria. Blue, phalloidin; red, collagen 1; green, nuclei (4',6-diamidino-2-phenylindole). B: collagen I expression as percent fractional area. Values are means ± SE from 10-wk-old male and female and 30-wk-old female TNF1.6 and control mice. L, left atrium; R, right atrium; Solid bars, TNF1.6; open bars, control. *P < 0.001; P < 0.02. No differences were observed between left and right atria for any given sex/age/genotype combination.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Representative recordings of force developed by isolated LA from 6- to 9-mo-old female control (WT, A) and TNF1.6 (TG, B) mice. Preparations were electrically stimulated at 1 or 6 Hz in the presence of 2 mM extracellular Ca2+ at 36°C. Right and left scale bars show total developed force (DF) and DF normalized to calculated cross-sectional area, respectively. Note differences in force scales between A and B and larger frequency-dependent reduction of DF in TNF1.6 versus control. Also, note the increase in basal tension in the TG atria.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Arrhythmias in TNF1.6 TG mice. A: telemetry traces from a 5-mo-old male control (top) and TNF1.6 mouse (bottom). Note clear P wave, QRS complex, and ST segment in these mice in sinus rhythms. B: atrial arrhythmias, including a run of a supraventricular tachycardia at a rate of 840 beats/min (top) and an irregularly irregular rhythm without P waves that appears to be atrial fibrillation (bottom).

In vivo electrophysiological testing. During in vivo electrophysiological testing, TNF1.6 mice had prolonged QT and HV intervals and more horizontal cardiac electrical axes (Tables 3 and 4). Young male TNF1.6 mice also had prolonged PR intervals compared with male controls, whereas older female TNF1.6 mice had impaired AV nodal (AVN) conduction with longer AVN Wenckebach periodicity and longer effective and functional refractory periods (Table 3). As expected, isoproterenol shortened RR, PR, and SNRTs as well as AVN Wenckebach and refractory periods in all mice. The differences in electrophysiological parameters between TNF1.6 and control mice persisted after -adrenergic stimulation (Table 3). During atrial and ventricular premature stimulation, there was no conduction discontinuity or jump noted in any transgenic or control mice.

View larger version (19K): [in this window] [in a new window]   Fig. 5. A: atrial reentrant tachycardia at a cycle length of 54 ms, with variable atrioventricular conduction induced by a single atrial extrastimulus in a male TNF1.6 mouse. B: surface and intracardiac tracings in sinus rhythm from a older female transgenic mouse. For both panels, top tracing represents lead I on the surface electrocardiogram. Middle and bottom tracings represent intracardiac (IC1 = HBE-p and IC2 = HBE-d) electrograms. A, atrial intracardiac deflection; V, ventricular intracardiac deflection; H, His deflection on intracardiac tracing; P, P wave; QRS, QRS complex on surface ECG.

Optical mapping of action potentials and programmed stimulation of arrhythmias in atria. We used the voltage-sensitive dye di-4ANEPPS to record action potentials from a 4 mm x 4 mm area on the epicardial surface, including the left atrium and ventricle of Langendorff-perfused isolated mouse hearts (Fig. 6A). Atrial APD at 75% repolarization (APD₇₅) and refractory periods were similar in TNF1.6 transgenic (13 ± 0.3 ms, n = 4 and 26 ± 4 ms, n = 6) and control hearts (12 ± 0.6 ms, n = 4 and 25 ± 3 ms, n = 4). Single premature atrial stimuli elicited slow atrial conduction and reentrant atrial tachycardias at rates up to 1,500 beats/min in 6 of 6 TNF1.6 transgenic but not 4 of 4 control hearts (Fig. 6, B and C). Isochronal maps and cinegraphic loops during the atrial tachycardias showed reentrant atrial activation with variable AV block, leading to the irregularly irregular ventricular rhythm on the surface ECG suggestive of atrial fibrillation.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Optical mapping of atrial action potentials and arrhythmias. A: simultaneous optical maps of the atrium (Atria, top 2 channels) and the ventricle (Vent, bottom 2 channels) during atrial pacing of a control heart (cycle length = 250 ms). Note that the ventricular signals are weakly visible in the atrial channels due to depth resolution. B: induction of an atrial tachycardia in a heart from a TNF1.6 TG mouse by a premature atrial stimulus (S2). Note that the cycle length of the atrial tachycardia is 40 ms, yielding an atrial rate of 1,500 beats/min, and that there is variable conduction to the ventricle leading to an irregularly irregular ventricular rate suggestive of atrial fibrillation. C: activation map of the last control S1 beat and during the S2 stimulus that induces the atrial tachycardia. Isochronal lines are 1 ms apart. Note that activation normally spreads across the paced atrium in <4 ms and that marked slowing and anisotropy of conduction are present during the premature beat.

Ca_i²⁺ activity in electrically stimulated atrial myocytes. Figure 7 shows representative examples of Ca_i²⁺ transients during 0.5-Hz electrical stimulation in left atrial myocytes isolated from hearts of 3-mo-old male TNF1.6 mice, from 6- to 9-mo-old female TNF1.6 mice, and from control littermates. Pooled data from these experiments are summarized in Table 5. Diastolic Ca_i²⁺, indexed by fura 2 ratio was not different among the compared groups. Relative to young male control myocytes, older female control myocytes displayed a small but statistically significant reduction in the amplitude, the maximal rate of rise (+dCa_i²⁺/dt_max) and decline (–dCa_i²⁺/dt_max) of the Ca_i²⁺ transient, and a longer transient duration (time to 50% decline, TD_50%). Compared with female control myocytes, female TNF1.6 myocytes displayed a significant reduction in the amplitude, the +dCa_i²⁺/dt_max and –d Ca_i²⁺/dt_max of the Ca_i²⁺ transient, and prolongation of the TD_50%. Compared with male control myocytes, male TNF1.6 myocytes displayed similar abnormalities. Compared with the younger TNF1.6 males, the Ca_i²⁺ transients in myocytes from older TNF1.6 females showed a significantly larger +dCa_i²⁺/dt_max but a longer TD_50%. The reduced amplitude of the Ca_i²⁺ transient and its +dCa_i²⁺/dt_max suggest a decrease in the SR Ca²⁺ release flux (42, 43), whereas the decrease in the –dCa_i²⁺/dt_max and its TD_50% indicate impaired SR Ca²⁺ uptake (32). In summary, these results show a significant impairment of the Ca_i²⁺ transient amplitude and kinetics in TNF1.6 versus control myocytes in age- and sex-matched groups. Changes in the configuration of the Ca_i²⁺ transients in TNF1.6 versus control atrial myocytes (Fig. 7, Table 5) are similar to those reported in ventricular myocytes isolated from failing human hearts and experimental animal models of CHF, including isolated ventricular myocytes from mice overexpressing TNF- (15, 20, 21, 27, 33).

Spontaneous Ca_i²⁺ activity in Ca²⁺-overloaded atrial myocytes. Figure 8 illustrates representative experiments in atrial myocytes from male control and TNF1.6 mice, demonstrating the occurrence and configuration of spontaneous Ca_i²⁺ transients, dubbed as "after transients," during a 30-s rest following Ca_i²⁺ overload (3). Ca²⁺ overload was induced by 30 s stimulation at 4 Hz and confirmed by a large increase in the diastolic versus resting fura 2 ratio (Fig. 8, A and B, left), comparable in magnitude to the amplitude (systolic-diastolic) of the electrically stimulated Ca_i²⁺ transients in control myocytes (Fig. 7, left). The after transients observed in the present experiments (Fig. 8, A and B, right and Table 5) fall into two distinct categories. One type of the after transients displayed smaller amplitudes and 5- to 8-fold smaller +dCa_i²⁺/dt_max relative to electrically evoked Ca_i²⁺ transients (Figs. 7 and 8 and Table 5) and, therefore, most likely reflected spontaneous Ca_i²⁺ "waves." The other type of after transients (e.g., right-most Ca²⁺ signal in Fig. 8B) was biphasic (i.e., started as a Ca_i²⁺ "wave" that transformed into a large, rapid Ca_i²⁺ transient comparable to electrically stimulated Ca_i²⁺ transients). The latter type of after transient most likely reflects synchronized SR Ca²⁺ release coordinated by a spontaneously triggered action potential (8, 29, 40, 46). Pooled data from these experiments showed that the total number of spontaneous Ca_i²⁺ transients was approximately three times more frequent in TNF1.6 versus control myocytes in both age/sex groups (Table 5). This reflected a 6- to 12-fold increase in the occurrence of the rapid, large after transients in the TNF1.6 versus control atrial myocytes and a not statistically significant 60% increase in the occurrence of Ca_i²⁺ waves. Taken together, these results show that atrial myocytes isolated from failing hearts overexpressing TNF- are more prone to the occurrence of spontaneous SR Ca²⁺ release due to intracellular Ca²⁺ overload and that the delayed afterdepolarizations (DADs) that result are more likely to reach the threshold for triggering spontaneous action potentials (3, 29, 40). The spontaneous SR Ca²⁺ releases and DADs can then induce triggered or reentrant arrhythmias through heterogeneity of repolarization and refractoriness, modulation of voltage inactivation of L-type Ca²⁺ channels during the action potential plateau, and/or repetitive generation of spontaneous action potentials (17, 29, 45).

SR Ca_i²⁺ content in atrial myocytes. Experiments using caffeine showed an 30% reduction in steady-state SR Ca²⁺ content in female TNF1.6 versus control atrial myocytes (0.21 ± 0.02 vs. 0.29 ± 0.03 fura 2 ratio units; P < 0.05; Fig. 9). These results are consistent with impaired SR Ca²⁺ uptake and/or Ca²⁺ loading and parallel the reduced amplitude and kinetics of the electrically stimulated Ca_i²⁺ transients (Figs. 7 and 9; Table 5). The findings are presumably due to impaired expression and/or function of sarco(endo)plasmic reticulum Ca²⁺-ATPase and/or the SR Ca²⁺ release channel/ryanodine receptor in the failing heart (8, 34). Defective regulation of the ryanodine receptor and increased diastolic Ca_i²⁺ may also underlie the more frequent occurrence of spontaneous Ca²⁺ transients in TNF1.6 versus control myocytes after rapid pacing (Fig. 8).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Arrhythmias remain a major source of morbidity and mortality in heart failure of both ischemic and nonischemic etiologies (49). Atrial arrhythmias such as atrial fibrillation or flutter are very common and constitute a significant problem to heart failure patients. In this study, we have shown that heart failure mice overexpressing the cytokine TNF- have a higher susceptibility to spontaneous and induced atrial arrhythmias with a higher risk in the males and that this increased predilection to developing atrial arrhythmias appears to be related to altered atrial morphology with fibrosis, abnormalities in Ca²⁺ handling with Ca²⁺ overload, and increased automaticity at the cellular level.

Atrial structural and functional abnormalities in TNF1.6 mice. Abnormalities in the atrial structure and function of the TNF1.6 mice include severe hypertrophy, fibrosis, contractile failure, altered force-frequency relationship, reduced amplitude and kinetics of electrically evoked Ca_i²⁺ transients, and a higher propensity to spontaneous SR Ca²⁺ release. Atrial structural and mechanical changes can predispose to arrhythmias. Atrial fibrosis and collagen III deposition have been implicated in the pathogenesis of atrial fibrillation in both human (4) and animal studies (6, 9, 31, 41, 47). The degree of fibrosis was associated with an increase in the expression of extracellular matrix proteins in patients with versus without atrial fibrillation, regardless of whether it is paroxysmal or chronic (9) In experimental heart failure, interstitial fibrosis is associated with significant local conduction abnormalities and likely helps to stabilize reentry and perpetuate atrial fibrillation or flutter. Also, atrial arrhythmias are more likely to occur in atria damaged by increased fibrosis and in atria stretched as a result of left ventricular hypertrophy or heart failure (10, 36, 37).

Role of calcium in atrial arrhythmia susceptibility in TNF1.6 mice. We have previously shown that mice overexpressing TNF- have longer ventricular APDs, no increased dispersion of refractoriness between apex and base, elevated diastolic and depressed systolic calcium transients, and prolonged Ca²⁺ transients compared with their littermate controls (33). In addition, premature ventricular beats exhibit diminished ventricular action potential amplitudes and conduct in a slow, heterogeneous manner in the TNF1.6 compared with control mice. Lowering extracellular Ca²⁺ normalizes conduction and prevents inducible ventricular arrhythmias in the transgenic mice. In the present study, our goal was to identify the determinants of higher susceptibility of the TNF1.6 mice to atrial arrhythmias and to compare the mechanisms to those underlying the ventricular arrhythmias.

It is well established that altered intracellular Ca²⁺ handling in the heart, including decreased peak systolic Ca²⁺, elevated diastolic Ca²⁺, and prolongation of the Ca²⁺ transient are present in the ventricle during heart failure (8, 18). These abnormalities of Ca²⁺ handling are associated with decreased expression of the SR Ca²⁺-ATPase (SERCA2a), variable decreases in phospholamban, leakiness of the ryanodine receptor, and increased expression of the Na⁺/Ca²⁺ exchanger and may contribute to ventricular arrhythmias (2). The results of our study support a role for similar abnormalities in Ca²⁺ handling in the genesis of atrial arrhythmias. The slow decay of the Ca_i²⁺ transient (Fig. 7, Table 5) and the increase in diastolic Ca_i²⁺, especially at rapid heart rates, increase the likelihood of spontaneous SR Ca²⁺ release (Fig. 8, Table 5), DADs, and DAD-mediated triggered arrhythmias (3, 17, 29, 45). This sequence of events is well established as a cause of ventricular tachycardias and can also represent the initial trigger for the atrial arrhythmia that can eventually perpetuate itself through reentry.

APD is prolonged in ventricles and ventricular myocytes from TNF1.6 mice (33). Whereas we cannot exclude prolongation of the terminal phase of the action potential, we did not identify prolongation of APD₇₅ in atria using optical mapping. This finding suggests that ion channel remodeling differs in the atria and ventricles and that APD prolongation is not necessary for arrhythmia susceptibility in this model.

Increased automaticity and triggered activity in TNF1.6 mice. Clinical atrial fibrillation originating from the posterior left atrium and pulmonary veins exhibits features of enhanced automaticity and triggered activity during its initiation phases (12). Our data shows increased spontaneous Ca²⁺ activity of two kinds: small Ca²⁺ waves and the larger synchronized Ca²⁺ transients probably representing spontaneous synchronized release of Ca²⁺ from the SR. The larger Ca transients likely produce DADs that may reach the threshold level for action potential initiation, which can be the triggers of arrhythmias. Thus abnormalities in calcium handling may lead to arrhythmias via automaticity and triggered activity.

Gender difference in arrhythmia susceptibility among TNF1.6 mice. Gender-related effects on the occurrence of certain types of arrhythmias have been appreciated for many years (1, 7, 11, 16, 30, 44). Based on epidemiological evidence in a CHF population, women have a better survival than men (19, 35). Recently, gender differences in survival have been demonstrated in the TNF1.6 heart failure mouse model, with affected female mice out living the affected male mice (25, 28).

In the present study, the affected male transgenic mice exhibited a more severe phenotype in terms of the induced cardiomyopathy and the accompanying electrophysiological abnormalities. Affected mice exhibit abnormalities of infraHisian conduction as well as ventricular repolarization compared with their age- and gender-matched controls, both before and after -adrenergic stimulation. These findings point to the intrinsic nature of these cardiac abnormalities, which are not affected by autonomic manipulation. Also, the close correlation of these findings with the cardiac electrical axis on the electrocardiogram and the degree of hypertrophy as measured by the heart-to-body weights ratio further supports the concept that the electrophysiological abnormalities are related to structural changes intrinsic to the heart. As the degree of myopathy progresses in the affected older female mice and young male mice, the heart becomes more globular and the electrical axis shifts to a more horizontal position. In addition, electrophysiological abnormalities worsen to involve further the distal conduction system and ventricular repolarization. These abnormalities may contribute to increased atrial pressure and worsen the vulnerability to spontaneous and inducible atrial arrhythmias.

The atrial arrhythmias seen in our study are primarily rapid reentrant atrial rhythms similar in nature to the atrial fibrillation and flutter seen in humans with advanced heart failure. The mode of induction with premature atrial stimuli both during in vivo and ex vivo testing as well as the cinegraphic propagation loops generated from the optical mapping data confirm the reentrant nature of these arrhythmias. The more severe phenotype seen in the male transgenic mice compared with the females is probably not a primary electrical phenomenon but a reflection of a more severe myopathy. Similarly, the prolongation of PR, HV, and QRS intervals correlates strongly with the degree of cardiac hypertrophy as measured by the heart-to-body weight ratio and are probably secondary to stretching and/or fibrosis of the specialized cells of conduction, primarily in the infraHisian region.

Mouse as a model for human arrhythmias. Many structural and electrophysiological differences exist between the mouse and human heart, including differences in size, heart rate, and various ionic channels. In addition, the single-cell studies in mouse atria were not performed at physiological temperatures. Thus our findings in the mouse should be interpreted with caution. The slower basal heart rate of the TNF1.6 mice compared with controls during ambulatory monitoring is one example. However, the TNF1.6 mice do develop many of the structural and biochemical findings present in human heart failure. Here, we show that these mice also develop atrial arrhythmias and have used in vivo and ex vivo techniques, including optical mapping of isolated hearts and single myocytes with voltage- and Ca²⁺-sensitive dyes to dissect the mechanisms of these reentrant arrhythmias. Our findings implicate changes in structure and intracellular Ca²⁺ handling as critical to the genesis of atrial arrhythmias. Further exploration of the pathways that couple these changes to atrial arrhythmias is warranted.

In conclusion, our results demonstrate the presence of severe structural and electrophysiological abnormalities in transgenic mice with cardiac-specific TNF- overexpression. These mice are highly susceptible to developing atrial arrhythmias in advanced stages, which can be related to a direct overexpression of TNF- in the atria and/or secondary to the hemodynamic effects of failing ventricles. This susceptibility to atrial arrhythmias seems to be related to atrial fibrosis, slowing of action potential propagation of premature beats, and altered calcium handling in atrial myocytes. The susceptibility to atrial arrhythmias can be reversed by modifying the external Ca²⁺ concentration in vitro, although we cannot distinguish whether the beneficial effects are due to changes in calcium handling or changes in atrial electrical conduction. These electrophysiological abnormalities are more pronounced in affected males and progress with aging. All these features mimic the characteristics of cardiomyopathy in humans and confirm the TNF1.6 mouse as a useful model for the study of atrial arrhythmias in heart failure. Its use in the future will include studying the effects of various therapeutic techniques on the progression or reversal of various abnormal morphological and electrophysiological parameters. As an example, mating the TNF1.6 mice with lines genetically targeted to alter atrial fibrosis and remodeling may be informative. In addition, the mice may be a valuable system in which to test novel pharmaceutical agents and genetic therapies.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported in part by American College of Cardiology Career Development Award in Arrhythmia for the year 2000 (to S. Saba) and by National Heart, Lung, and Blood Institute Grants HL-58030, HL-66096 (to B. London), HL-60032 (to C. McTiernan), and HL-59614 (to G. Salama).

	FOOTNOTES

 

Address for reprint requests and other correspondence: B. London, Cardiovascular Institute, Univ. of Pittsburgh, 200 Lothrop St., Scaife S572, Pittsburgh, PA 15213 (e-mail: londonb{at}upmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* S. Saba and A. M. Janczewski contributed equally to this study.

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