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Am J Physiol Heart Circ Physiol 282: H842-H849, 2002. First published October 11, 2001; doi:10.1152/ajpheart.00240.2001
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Vol. 282, Issue 3, H842-H849, March 2002

A possible role for atrial fibroblasts in postinfarction bradycardia

Andre Kamkin1,3, Irina Kiseleva1,3, Kay-Dietrich Wagner1, Alexander Pylaev1, Kate P. Leiterer2, Heinz Theres2, Holger Scholz1, Joachim Günther1, and Gerrit Isenberg3

1 Institute of Physiology and 2 Clinic of Internal Medicine I, Humboldt-University, Charité, 10117 Berlin; and 3 Department of Physiology, Martin-Luther-University of Halle, 0697 Halle/Saale, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Atrial fibroblasts are considered to modulate the contractile activity of the heart in response to mechanical stretch. In this study we examined whether atrial fibroblasts are possibly involved in bradyarrhythmia, which is a severe complication after myocardial infarction. For this purpose, transmembrane electrical potentials were recorded in cardiac fibroblasts near the sinoatrial node from sham-operated rats and from rats with myocardial infarction. Twenty days after infarction due to coronary artery ligation, the right atrial tissue weights and the sensitivity of the fibroblast membrane potential to mechanical stretch correlated positively with the infarct size. Cardiac growth was enhanced, but the stretch sensitivity and the resting membrane potential of the atrial fibroblasts declined between 8 and 30 days after infarction. The frequency of spontaneous atrial contractions was significantly reduced 8 days after myocardial infarction and recovered in parallel with the membrane potential of the fibroblasts. These findings suggest that changes in the susceptibility of atrial fibroblasts to mechanical stretch may contribute to bradyarrhythmia during postinfarct remodeling of the heart.

cardiac hypertrophy; stretch-activated ion channels; mechanotransduction; membrane potential; gadolinium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BRADYARRHYTHMIA IS A critical complication that occurs in a significant minority of patients with acute myocardial infarction (MI) (reviewed in Ref. 2). The molecular mechanisms that underlie the depression of heart rates in the (post)ischemic myocardium are incompletely understood but may involve functional and structural damage to the cardiac pacemaker cells and the conduction system (1). We and others (12, 14, 16, 17) have shown previously that the frequency of spontaneous contractions of the nonischemic heart is modulated by the electrical activity of atrial fibroblasts. Thus mechanical stretching hyperpolarized the resting membrane potential of atrial fibroblasts and decreased the frequency of spontaneous contractions of isolated right atrial tissue preparations from rat hearts (13). The latter phenomenon, which is thought to play a role in the adaptation of the heart to changing work load, is commonly referred to as mechanoelectrical feedback (18, 19). Furthermore, lateral compression of the fibroblasts during systolic contraction of the cardiac myocytes may depolarize the membrane potential, thereby eliciting so-called "mechanically induced potentials" (MIPs) (14, 15). The identity of the putative signal that is generated by the atrial fibroblasts in response to physical stretch has remained unknown. It is also unclear as to how the predicted signal is transmitted from the fibroblasts to the pacemaker cells in the sinoatrial node.

Among other mechanisms, cardiac remodeling after MI involves proliferation of atrial fibroblasts and changes in the electrical properties of these cells. Thus atrial fibroblasts in the postischemic myocardium acquired a myocyte-like phenotype (7), and the increased resting membrane potential of these cells exhibited enhanced susceptibility to mechanical stretch (15). Hyperpolarization of the membrane potential of atrial fibroblasts in the nonischemic myocardium was associated with reduced contractile activity and arrhythmia (14, 16, 17). These findings raise the interesting possibility that disturbed electrical function of atrial fibroblasts after MI contributes to bradyarrhythmia during postinfarct remodeling of the heart. In a first approach to test this hypothesis we examined the influence of mechanical stretch on the resting membrane potential of atrial fibroblasts at varying infarct sizes and different time points after MI. The results from the electrophysiological recordings were compared with the frequency of spontaneous contractions of rat hearts in vivo and of right atrial tissue preparations in vitro.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of myocardial infarction. The investigation conforms with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, 1996). One hundred twenty-one male Wistar rats (age 10-15 wk, body weight 200-350 g) were anesthetized by intraperitoneal injection of 75 mg/ kg ketamine hydrochloride (Ketanest; Sanofi, Düsseldorf, Germany) mixed with 7.5 mg/kg xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany). Experimental MI was induced as described previously (15). Briefly, the heart was exteriorized after left-sided thoracotomy, and the left coronary artery (LAD) was ligated ~3 mm and 5 mm downstream from its origin to cause large and small size infarcts, respectively. Successful ligation was verified by the occurrence of arrhythmia and S-T segment elevation in the electrocardiogram (ECG). The thorax was closed after repositioning of the heart. After the rats recovered from anesthesia, 20 mg gentamycin sulfate (Refobacin; Merck, Darmstadt, Germany) were applied subcutaneously to each rat. Cardiac surgery caused a postoperative mortality within the first 24 h of ~40% with the large size infarcts and <6% with small infarcts. Sham-operated (SO) rats underwent the same surgical procedure except that the "ligation" was placed beside the coronary artery. The mortality was zero in this group.

Grouping of the hearts according to the infarct size and the time after infarction. The rats were anesthetized with ether and decapitated after ECG recordings were made. The hearts were quickly excised, and the infarct size (IS) was determined by the ratio of the wet weights of the left ventricular scar tissue to the total left ventricular tissue (free wall and septum). In a first series of experiments, the hearts were removed 20 days after surgery and grouped according to the IS. Proximal and distal ligation of the LAD resulted in ISs of ~40 and 16%, respectively. A third group of hearts developed no macroscopically visible infarction despite arrhythmia and S-T elevation after coronary artery ligation (group IS0 = nonvisible). To facilitate statistical analysis, we used only hearts with <5% difference from the average infarct size in each group. For this reason, ~40% of the hearts were discarded and the remaining preparations were assigned to one of the following groups: IS40 (IS of 40.2 ± 0.3%, n = 9), IS16 (IS of 16.5 ± 0.6%, n = 10), and IS0 (nonvisible infarction, n = 8). In a second set of experiments, the membrane potential of rat atrial fibroblasts was analyzed at different time points after MI. For this purpose, hearts with 16% IS were studied 8 days (MI8, n = 6), 20 days (MI20, n = 10), and 30 days (MI30, n = 10) after distal ligation of the LAD. SO rats (n = 15) at 20 days after surgery served as negative controls.

Atrial tissue preparations and bath solutions. Right atria were removed and dissected in an oxygenated physiological salt solution. A sharp metal stamp (width, 4 mm; length, 8 mm) was oriented in parallel to the visible texture of the atrial fibers and used to cut out an atrial tissue specimen including the leading pacemaker site. After isolation, the preparation was put in a thermostatically controlled (37°C) perfusion chamber with the endocardial surface facing up. The perfusate contained (in mM) 118 NaCl, 2.7 KCl, 1.2 CaCl2, 1.2 MgSO4, 2.2 NaH2PO4, 25 NaHCO3, and 5 glucose and was bubbled with a 5% CO2-95% O2 mixture to adjust pH to 7.4 (3). To avoid precipitation of solutes in the presence of gadolinium (GdCl3, 40 µM), these experiments were performed with the following salt solution (in mM), which was gassed with pure oxygen (26): 137 NaCl, 5.4 KCl, 1.0 CaCl2, 0.5 MgSO4, 5.0 HEPES, and 5 glucose (37°C, pH 7.4). No significant differences of the membrane potentials of the atrial fibroblasts and their responses to mechanical stretch were observed between the two bath solutions.

Mechanical recordings and stretch of the atrial tissue preparations. The preparations were fixed horizontally with clips from tungsten wire between the force transduction system (Plugsys 603; Hugo Sachs Elektronik) and a micrometer that was used for the adjustment of preload and stretch. With the baseline preload set to 1 mN, the developed active force had an amplitude of ~0.3 mN. Long-lasting mechanical stretch was applied in parallel to the cardiac muscle fibers to simulate changes in right atrial pressure and atrial dilatation, respectively. Similar to previous reports (10, 15), a preload of 1 mN caused lengthening of the atrial preparations from SO and MI rats by ~6%.

Stretch, i.e., steplike increments of the length of the right atrial tissue preparations, was applied slowly (~0.1 mm/s) to avoid dislocation of the recording microelectrode. The adjusted resting forces were held constant for ~3 s. Even though this stretch protocol did not reflect the beat-to-beat variations of the right atrial filling pressure, it presumably simulated the longer lasting changes in atrial chamber dilatation after MI.

Electrophysiological recordings. Membrane potentials of atrial fibroblasts were recorded with a floating microelectrode as described earlier (14). Briefly, a short Ag/AgCl pin was connected to the headstage of the amplifier via platinum-iridium wire (25 µm diameter). This pin was partly inserted into a glass microelectrode containing 2.5 M KCl that had a tip resistance between 10 and 20 MOmega . The reference micropipette was a short Ag/AgCl pin in a glass microelectrode containing 2.5 M KCl. Both electrodes were connected to a high-impedance input amplifier (BP1; Medico-Biological Industrial, Moscow, Russia) in current-clamp mode. The output signal of the amplifier was displayed on an oscilloscope (Nicolet Pro10; Nicolet) and a digital tape recorder (DTR-1802; Biologic). Because the microelectrode tip resistance was low (10-20 MOmega ) compared with the input resistance of the fibroblasts (~500 MOmega ), we did not compensate for the voltage drop across the microelectrode resistance caused by injection of negative and positive current pulses (usually 0.1 nA). Considerable differences in the resting membrane potentials of individual fibroblasts were observed between preparations from both SO and MI rats (-5 to -70 mV, Ref. 15). For better comparison of the results, the cells were polarized to a membrane potential of -30 mV before starting the experiments. We used only those recordings for statistical evaluation in which we could complete a full-length protocol with all steps of stretch and relaxation during stable electrical activity. A total number of ~230 cells from 58 different preparations were monitored. The recorded data were digitized, saved on a personal computer, and analyzed off-line.

Identification of atrial fibroblasts. The following criteria were applied to distinguish between membrane potentials recorded from atrial fibroblasts and cardiac myocytes. 1) The fibroblast resting membrane potentials were less negative (-22 ± 2 mV on average) than the resting potentials of the atrial myocytes (-88 ± 2 mV). 2) The input resistance of the fibroblasts was high (510 ± 10 MOmega ) compared with the input resistance of the cardiac myocytes (10-40 MOmega ). 3) Action potentials were never detected and could also not be induced by depolarizing and/or hyperpolarizing current pulses in atrial fibroblasts. 4) MIPs (see below) could be elicited only in fibroblasts but not in cardiac myocytes.

The recorded cells were labeled by intracellular microiontophoresis of Lucifer yellow (LY) through the recording microelectrode (14). Staining of individual cells was only detected when LY was injected into fibroblasts. In contrast, microiontophoresis of LY into cardiac myocytes caused transmembrane spreading of the dye due to gap junctional coupling of these cells. In some experiments colloidal gold particles were applied into the cells through the recording microelectrode. With the use of electron microscopy, the injected cells could be undoubtedly identified as fibroblasts (12). For this purpose, the microelectrode tip was cut and the atrial tissue (~1 mm2) was dissected with the microelectrode tip. The tissue specimens were fixed in 4% glutaraldehyde in Millonig's buffer at 4°C (pH 7.4) and postfixed in 1% osmium tetroxide in Millonig's buffer, dehydrated, and embedded in Epon 812. Ultrathin sections were stained with lead citrate and uranyl acetate according to Reynolds (20) and inspected with a Hitachi HU-11-2E electron microscope at magnifications between ×3.200 and ×31.000. As expected from previous studies (6), >90% of the nonmyocytes were identified as fibroblasts.

Statistics. All data are presented as means ± SE. The Levene test for inhomogeneity of variances was performed. Intergroup comparisons were done using a one-way ANOVA with the Bonferroni test as a post hoc test (SPSS statistical package; SPSS). The effect of GdCl3 on the membrane potential of the atrial fibroblasts was evaluated using the paired Student's t-test. Significance was assumed at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Morphology of right atrial tissue preparations from SO and infarcted rat hearts. Electron microscopy was performed to assess the morphological criteria of cardiac remodeling in right atrial tissue after myocardial infarction. Examination of tissue from SO rats revealed the typical composition of cardiomyocytes, fibroblasts, and rare collagen fibers (Fig. 1A). The somata and processes of the fibroblasts extended close to the surface of the myocytes (~250 nm distance), and thin processes anchored to the basement membranes of the fibroblasts formed visible contacts between both cell types. Similar to the sinoatrial region of rabbit hearts (5), specialized contacts between fibroblasts and myocytes such as gap junctions, tight junctions, and desmosomes could not be detected. In agreement with the results from morphometric studies (24), the fraction of fibroblasts was estimated to be ~25% in the right atrial tissue of SO rats, based on the characteristic resting membrane potential of these cells (Table 1). Twenty days after MI (16% IS), the right atrial tissue showed the typical signs of hypertrophy with accumulation of collagen fibers in the interstitial space (Fig. 1B). The degree of cardiac hypertrophy was estimated by comparing the wet weights of the left ventricles and the right atria, which were normalized to the body weights of the animals. As shown in Tables 1 and 2, enhanced right atrial and left ventricular growth correlated closely with both the IS and the time after MI. The fraction of fibroblasts identified by electron microscopy was approximately doubled 20 days after MI compared with SO. Accordingly, the microelectrode recordings suggested a ratio of the number of fibroblasts to the total number of cells of ~55% in the hypertrophied right atria.


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  		Fig. 1.   Electron micrographs of right atrial tissue from a sham-operated rat (A) and from a rat 20 days after myocardial infarction (B). M, myocytes; F, fibroblasts; SR, sarcoplasmic reticulum; mi, mitochondria; C, collagen; gj, gap junctions. Micrographs shown were obtained from tissue samples that had previously been used for microelectrode recordings. Note the accumulation of collagen fibers in the interstitial space and the absence of gap junctions, tight junctions, and desmosomes between the fibroblasts and cardiac myocytes. Scale bar, 2.5 µm.


                              
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  		Table 1.   Cardiac hypertrophy, active force, spontaneous contractile activity, and atrial fibroblast membrane potential in sham-operated rats and at different infarct sizes 20 days after coronary artery ligation


                              
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  		Table 2.   Cardiac hypertrophy, spontaneous contractile activity, and fibroblast membrane potential at different time points after myocardial infarction (16% infarct size)

Resting membrane potentials and mechanically induced potentials of atrial fibroblasts from SO and infarcted rat hearts. Intracellular recordings were performed on right atrial tissue specimens, which included the sinoatrial pacemaker region. With the mechanical preload set to 1 mN, the atrial fibroblasts from SO rats had a resting potential (Vm) of -22 ± 2 mV (Table 1), and their input resistance was 510 ± 10 MOmega (n = 71). Vm of the atrial fibroblasts increased with the IS from -25 ± 2 mV at nonvisible infarctions to -45 ± 2 mV at 40% IS. The fibroblast membrane potential reached a maximum value (-41 ± 3 mV) 8 days post-MI (16% IS) and decreased to -28 ± 3 mV within 30 days after coronary artery ligation (Table 2).

Spontaneous contractions of the right atrial in vitro preparations were associated with depolarization of the fibroblasts known as MIPs. The MIPs could be distinguished from the action potentials of the cardiac myocytes by their slower depolarization rates and the duration of ~100 ms. The amplitudes of the MIPs were dependent on the resting membrane potential of the fibroblasts. For example, the fibroblast shown in Fig. 2A has a MIP amplitude of 15 mV at a resting membrane potential of -29 mV. When Vm was adjusted to -50 mV by injection of hyperpolarizing current pulses, the amplitude of the MIPs increased to 23 mV (Fig. 2A). MIPs were no longer observed after depolarization of the fibroblast to the reversal potential (Erev) of -4.5 mV. Application of mechanical stretch by raising the preload to 2.5 mN enhanced the active force by ~20% and hyperpolarized the resting membrane potential to -55 mV (Fig. 2B). As a consequence, the MIP amplitude increased and Erev shifted to more negative values (-9 vs. -4.5 mV at a preload of 1 mN). A detailed analysis of the effect of changes in resting force on Vm and MIP amplitudes of atrial fibroblasts from SO rats is given in Fig. 3. Without preload, Vm was -12.0 ± 3 mV (Fig. 3, open circles), and mechanical stretch hyperpolarized the cells along an S-shaped curve that was fitted with Vm = Vm,o + Delta Vm,max/{1 + exp[-(F - F0.5)/slope]}, where F0.5 is the force for half-maximal effect, slope represents the stretch sensitivity, Delta Vm,max is the maximal amplitude of stretch-induced hyperpolarization, and Vm,o is the resting potential in the absence of stretch, to yield a slope of -41 mV/mN. The effect of stretch on Vm was half-maximal at F0.5 = 1.4 mN, and maximal hyperpolarization (Delta Vm,max = -62 mV) of the fibroblasts from Vm,o =-12 to -74 mV was obtained at a resting force of 3 mN. Similar to the changes in Vm, the MIP amplitudes (Fig. 3, solid circles) increased with the resting force along an S-shaped curve with a slope of 38 mV/mN. Half-maximal amplitude of the MIPs was observed at 1.4 mN resting force, and the largest MIPs (65 mV) occurred at 3 mN. Stretching of the preparations to resting forces beyond 3 mN reduced the MIP amplitudes (Fig. 3). With the preload set to 1 mN, the stretch sensitivity of the MIPs was 2.8 mV/0.1 mN. The amplitudes of the MIP increased along with the resting membrane potential of the atrial fibroblasts from 17 ± 4 mV at nonvisible infarction to maximally 41 ± 3 mV at 40% IS (Table 1). The MIP amplitudes decreased in parallel with Vm between 8 and 30 days after MI (Table 2).


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  		Fig. 2.   Effect of changing resting membrane potential (RMP) on mechanically induced potentials (MIPs) in an atrial fibroblast from sham-operated rat. The preload was set to 1 mN in the experiment shown in A. Note that the amplitude of the MIPs increased upon injection of hyperpolarizing currents (downward arrows) and decreased with depolarizing currents (upward arrows). MIPs were blunted by adjusting the membrane potential of the fibroblast to the reversal potential (Erev) of -4.5 mV. Top trace indicates the resting force (RF) and the active force (AF) developed by rhythmic contractions of the right atrial tissue preparation. The current injections applied to adjust the membrane potential of the fibroblast to the desired values are indicated on the bottom trace. B: recording from the same atrial fibroblast as in A with the resting force set to 2.5 mN. Note that increasing preload hyperpolarized the resting membrane potential and increased the amplitude of the MIPs.



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  		Fig. 3.   Effect of changing resting force on the resting membrane potential (Vm) and the amplitude of MIPs in atrial fibroblasts from sham-operated rats. Open circles indicate Vm; dots symbolize the MIP amplitudes. The data were fitted with a sigmoidal curve Vm = Vm,o + Delta Vm,max/{1 + exp[-(F - F0.5)/slope]}, where F0.5 is the force for half-maximal effect, slope represents the stretch sensitivity, Delta Vm,max is the maximal amplitude of stretch-induced hyperpolarization, and Vm,o is the resting potential in the absence of stretch. For Vm, the fit yielded Vm,o = -12 mV, Vm,max = -62 mV, F0.5 = 1.4 mN, and slope = -41 mV/mN. For MIP, the fit yielded Vm,o = 8 mV, Vm,o + Vm,max = 65 mV, F0.5 = 1.4 mN, and slope = 38 mV/mN.

Interestingly, the susceptibility of the membrane potential of the atrial fibroblasts to mechanical stretch was enhanced after infarction. Thus increasing stretch by 0.1 mN hyperpolarized the atrial fibroblasts by -3 mV in SO rats and by -35 mV in the IS40 group (Fig. 4). Maximal stretch-dependent hyperpolarization at 0.3 mN stretching force was -78 mV with 40% IS (Fig. 4). The hyperpolarizing effect of physical stretch correlated inversely with the size of the infarctions. Thus maximal stretch-dependent hyperpolarization (Delta Vm) was -52 mV at 16% IS and -12 mV with nonvisible infarctions, respectively (Fig. 4). Enhanced sensitivity of Vm to mechanical stretch with increasing IS is also evident from the slopes in Fig. 4, which were -3 mV/0.1 mN (SO), -4.9 mV/0.1 mN (IS0), -17.5 mV/0.1 mN (IS16), and -32 mV/0.1 mN (IS40). Time-dependent changes of Vm were analyzed in more detail at 16% IS. As shown in Fig. 5, the sensitivity of Vm to stretch was maximal on day 8 after MI with a resting force of only 0.1 mN causing hyperpolarization of the membrane potential by -78 mV. Twenty and 30 days after coronary artery ligation the stretch sensitivity decreased to -17.5 mV/0.1 mN and -4.9 mV/0.1 mN, respectively.


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  		Fig. 4.   Changes of the resting membrane potential of atrial fibroblasts in response to mechanical stretch. Data shown were obtained from sham-operated rats (open circles, n = 15) and from rats with nonvisible infarction (solid circles, n = 8), 16% infarct size (IS) (triangles, n = 10), and 40% IS (squares, n = 9) 20 days after ligation of the coronary artery. Note that the sensitivity of Vm of the atrial fibroblasts to mechanical stretch increased with the IS from -3 mV/0.1 mN (sham operation) to -4.9 mV/0.1 mN (IS = 0%), -17.5 mV/0.1 mN (IS = 16%), and -32 mV/0.1 mN (IS = 40%), respectively. ++ P < 0.01 vs. sham operation. +++ P < 0.001 vs. sham operation.



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  		Fig. 5.   Changes of the resting membrane potential of atrial fibroblasts in response to mechanical stretch at different time points after myocardial infarction (MI). Shown are the results from sham-operated rats (open circles, n = 15) and from rats with 16% IS at 8 days (squares, n = 6), 20 days (triangles, n = 10), and 30 days (solid circles, n = 10) after MI. Note that the sensitivity of the membrane potential of atrial fibroblasts to physical stretch decreased with the time after infarction from -78 mV/0.1 mN at day 8 to -17.5 mV/0.1 mN at day 20, and to -4.9 mV/0.1 mN at 30 days post-MI. +++ P < 0.001 vs. sham operation.

Next we examined whether nonselective cation channels (NSCs) were possibly involved in the generation of MIPs by atrial fibroblasts. For example, activation of NSCs in response to mechanical stretch has been demonstrated in a variety of tissues including myocardial cells (11, 22, 23, 25, 27). As shown in Fig. 6A, gadolinium (GdCl3) used at a concentration of 40 µM to inhibit NSCs (26) markedly reduced the stretch-dependent hyperpolarization of the fibroblasts 20 days after infarction (IS16). Thus the stretch sensitivity of Vm was -17 mV/0.1 mN without and -9 mV/0.1 mN in presence of GdCl3. In addition to the changes of Vm in response to mechanical stretch, GdCl3 significantly reduced the MIP amplitudes at resting membrane potentials below -20 mV. This effect was further enhanced by hyperpolarizing Vm of the atrial fibroblasts (Fig. 6B). At normal Vm of -22 mV, the slope of the plots was -0.58 without gadolinium and -0.30 in the presence of GdCl3, suggesting that GdCl3 lowered the sensitivity of the MIPs to changes in the resting membrane potential of the fibroblasts.


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  		Fig. 6.   Effect of gadolinium, an inhibitor of nonselective cation channels, on the stretch sensitivity of the Vm (A) and MIP amplitude (B) of atrial fibroblasts. The tissue samples were obtained from rat hearts with 16% IS 20 days after coronary artery ligation (n = 12). The experiments were performed in the absence (open circles) and presence of gadolinium (40 µM, solid circles). Note that gadolinium (GdCl3) lowered the stretch sensitivity of Vm from -19 to -9 mV/0.1 mN and also significantly reduced the MIP amplitudes at resting membrane potentials below -20 mV. The experiments were performed with the preload set to 1 mN. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. controls without GdCl3.

Changes of the contractile activity after MI. Finally, we explored whether the observed changes in electrophysiology of the atrial fibroblasts may contribute to altered contractile activity of the myocardium after MI. For this purpose, the in vivo heart rates and the frequency of spontaneous atrial contractions in vitro were studied at varying IS and different time points after MI. Notably, increasing ISs were associated with depression of the heart rates 20 days after coronary artery ligation. Thus the mean frequency of myocardial contractions was 420 ± 6 beats/min in SO rats, 360 ± 8 beats/min in animals with nonvisible infarct, 270 ± 11 beats/min at 16% IS, and 240 ± 9 beats/min at 40% IS (Table 1). Similar to the heart rates in vivo, the frequency of spontaneous contractions of the isolated atrial tissue preparations decreased from 324 ± 5 beats/min in SO rats to 172 ± 8 beats/min at 40% IS (1 mN preload). The time-dependent changes of the contractile activity after MI were examined in more detail at 16% IS. As shown in Table 2, both the frequency of spontaneous contractions in vivo and in vitro increased between 8 and 30 days after infarction. Thus a striking correlation was seen between the resting membrane potential of atrial fibroblasts, its sensitivity to mechanical stretch, and the spontaneous contractile activity of the myocardium after MI.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Atrial fibroblasts are thought to act as "mechanosensors" that can modulate the electrical activity of the pacemaker cells in the heart. We have recently demonstrated that the resting membrane potential and MIPs of atrial fibroblasts are sensitive to physical stretch (14) and correlate with the IS (15). In this study we examined whether atrial fibroblasts possibly play a role in bradyarrhythmia, which is a critical complication during postinfarct remodeling of the heart. We report the following novel observations. 1) Hyperpolarization of the Vm of rat atrial fibroblasts is maximal 8 days post-MI and recovers, albeit incompletely, within 30 days after infarction. 2) Atrial fibroblasts are hyperpolarized by mechanical stretch, and this effect is attenuated with the time after MI. 3) The MIPs are sensitive to gadolinium and correlate closely with the changes of Vm after infarction. 4) Depression of contractile activity of the myocardium follows the hyperpolarization of the fibroblast membrane potential after MI. These findings raise the possibility that changes of the membrane potential of atrial fibroblasts contribute to postinfarct bradycardia.

Interestingly, the sensitivity of Vm of the fibroblasts to mechanical stretch changed in a characteristic fashion after MI. Thus the maximal response of atrial fibroblasts to physical stress was seen 8 days after infarction and then decayed within 30 days post-MI. In contrast, atrial hypertrophy developed more slowly and became statistically significant 30 days after infarction. It appears less likely, therefore, that modulation of the susceptibility of Vm to physical stress was due to changes in the tension and/or length of the fibroblasts, which may result from atrial hypertrophy. Instead, our findings suggest a more specific effect of (post)ischemic remodeling on the electrical membrane properties of atrial fibroblasts. This conclusion is supported by the positive correlation between the IS and the stretch sensitivity of the atrial fibroblasts.

The molecular mechanisms that underlie the changes in Vm of the atrial fibroblasts during physical stress are yet unknown. Fibroblasts respond to local mechanical stretch with an increase in the cytosolic Ca2+ concentration, which results from both transmembrane Ca2+ influx and Ca2+ release from intracellular stores (4, 8). Consistent with a role for calcium ions in the mechanoelectric coupling, we have recently reported that hyperpolarization and mechanically induced potentials in atrial fibroblasts can be blocked by keeping the intracellular Ca2+ concentration low (14). Here we show that the MIPs are sensitive to gadolinium, suggesting a mechanism of transmembrane influx through NSCs to be involved (25). Notably, coordinated changes of Vm and MIP amplitudes of the atrial fibroblasts were observed after myocardial infarction. This close temporal correlation was expected based on the assumption that the amplitude of the MIPs is determined primarily by the force (Vm - Erev), which drives cations through NSCs at constant Erev of approximately -5 mV. More precisely, the amplitude of the MIPs is determined by (Vm - Erev) only under conditions where the conductance of NSCs is large compared with the conductance of other ion channels. Accordingly, the amplitude of the MIPs did not linearly increase with (Vm - Erev) when a large number of additional channels were activated as expected during maximally enhanced stretch (Fig. 3) or when NSCs were blocked by the use of gadolinium (Fig. 6B).

The question arises as to how the changes of Vm and MIPs of the atrial fibroblasts may affect the spontaneous contractile activity of the heart. The efficiency by which the membrane potential of the fibroblasts can modulate the heart rates is expected to increase during gap junctional coupling between the fibroblasts and the pacemaker cells in the sinoatrial node as suggested by Kohl et al. (16). However, we could not detect differences in the input resistance between atrial fibroblasts from SO and infarcted rat hearts. Furthermore, electron microscopy did not provide evidence for gap junctional coupling between atrial fibroblasts and myocytes as reported from coculture experiments (21). Thus it is tempting to speculate that electrical cross-talk between atrial fibroblasts and sinoatrial nodal cells involves capacititative coupling of the cell membranes. In fact, capacitative coupling occurs in most tissues with time constants of ~1 ms (9), which is sufficiently fast to allow efficient signal transfer from the atrial fibroblasts to the cardiac pacemaker cells. On the other hand, our findings do not allow us to exclude the possibility that atrial fibroblasts release a humoral signal in response to mechanical stretch, which can modulate the spontaneous contractile activity of the heart.

In summary, atrial fibroblasts are considered to sense atrial filling and wall stress through changes in their resting membrane potential (17). In this study we demonstrate for the first time that altered heart rates after MI correlate closely with hyperpolarization of the resting membrane potential and mechanically induced potentials in atrial fibroblasts. Even though a causative role for the observed changes in the fibroblast membrane potential remains to be established, we propose that enhanced sensitivity of atrial fibroblasts to mechanical stretch contributes to bradycardia during postinfarct remodeling of the heart.


    ACKNOWLEDGEMENTS

This study was supported by the Alexander von Humboldt-Stiftung (Germany). A. Kamkin was a fellow of the Alexander von Humboldt-Stiftung (Germany) and the Deutsche Forschungsgemeinschaft (DFG). I. Kiseleva received travel grants from the Humboldt University of Berlin and the DFG.


    FOOTNOTES

Address for reprint requests and other correspondence: K.-D. Wagner, Johannes-Müller-Institute of Physiology, Humboldt-University, Charité, Tucholskystrasse 2, 10117 Berlin, Germany (E-mail: kay-dietrich.wagner{at}charite.de).

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.

10.1152/ajpheart.00240.2001

Received 12 March 2001; accepted in final form 8 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(3):H842-H849
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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