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1Davis Heart and Lung Research Institute, 2Ohio State University Biophysics Program, 3College of Pharmacy; and 4Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio
Submitted 31 October 2005 ; accepted in final form 25 May 2006
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ABSTRACT |
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17 mo); myocytes from age-matched rats served as controls. Inward IK1 was significantly reduced at both 8 and 17 mo in SHHF rats compared with controls. There was a reduction in inward IK1 due to aging in the controls only at 17 mo. We found a significant increase in If at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in If. Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an If blocker (10 µM zatebradine). Increased If during hypertension alone or combined increases in If with reduced IK1 during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.
aging; pacemaker current; inward rectifier potassium current; abnormal automaticity
Spontaneous hypertensive heart failure (SHHF) rats develop hypertension at an early age. In contrast to spontaneously hypertensive rats (SHRs), the SHHF rats consistently develop reproducible, hypertensive heart failure in an age-dependent manner (26). Echocardiographic studies in the SHHF rat demonstrate left ventricular dysfunction (left ventricular ejection fraction <40%) at 17–18 mo of age (31). There is also evidence of progressive cardiac hypertrophy in the SHHF rats, evidenced as an increase in heart weights (36).
Hypertension and heart failure can increase the pacemaker current (If) in the ventricular myocardium (4–7). This can pathologically alter the diastolic phase of the action potential and enhance abnormal automaticity. In addition, aging has been shown to increase If density in both normal and hypertensive (SHR) rat ventricles (5).
Normally, the diastolic membrane potential is primarily regulated by the inward rectifier K+ current (IK1). IK1 is reduced in heart failure and, consequently, may contribute to increased excitability of the ventricle (30). We hypothesized that chronic hypertension and the resultant heart failure would lead to altered diastolic membrane currents, providing a substrate for abnormal excitability and automaticity. We measured IK1 and If in SHHF rats during the development of hypertension and heart failure in comparison to age-matched controls. Our observations suggest that changes in IK1 and If provide a mechanism for the initiation of ventricular arrhythmias during hypertension, heart failure, and aging.
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METHODS |
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Animals were deeply anesthetized with intraperitoneal injection of pentobarbital sodium, and the heart was rapidly removed. After the heart was flushed, it was weighed, and heart weight-to-body weight ratio was calculated. All animal procedures were approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
Voltage-clamp recordings. Ventricular myocytes were isolated by using a previously described method (3). Myocytes were stored at room temperature under an oxygen hood until studied. Only cells with clear striations and sharp margins were studied. All data were acquired within 8 h of cell isolation to minimize potential time-dependent changes in the currents.
Cells were placed in a dish (Cell Microcontrols, Virginia Beach, VA) and superfused with test solutions; the bath temperature was maintained at 35°C with a temperature controller (Cell Microcontrols). Solutions were changed with a six-port gravity flow system (
1 ml/min). Data acquisition was performed with pClamp software (version 8, Axon Instruments, Union City, CA) and an Axopatch 200A patch-clamp amplifier (Axon Instruments).
The amphotericin B-perforated, whole cell, patch-clamp technique was used for all recordings to minimize alterations in the intracellular milieu. Patch pipettes (2–5 M
) were used, and after seal formation, changes in the capacitative response to a –10-mV step were used to monitor perforation of the patch by amphotericin B. Series resistance compensation was applied (40–60%) to minimize voltage errors. Only cells with low access resistance (<20 M) that was stable (<20% change in series resistance) were included in the data analysis.
The bath solution contained (in mM) 134 NaCl, 1 MgCl2, 5 KCl, 5 HEPES, 1 CaCl2, and 5 D-glucose; pH was adjusted to 7.40 with NaOH. Nifedipine (2 µM) was added to the bath solution to block L-type calcium current. The pipette solution contained (in mM) 130 KCl, 5 MgCl2, 5 HEPES, and 5 EGTA; pH was adjusted to 7.2 with KOH.
A holding potential of –40 mV was used for all voltage-clamp experiments to inactivate sodium current. A series of 100-ms test potentials from –140 to +40 mV, with a 20-mV increment, were used to elicit IK1. The current was measured at the end of each 100-ms test potential. IK1 was defined as barium-sensitive current and was measured as the difference in current between that in the control solution and that in the bath solution with 2 mM BaCl2. If was measured after the addition of BaCl2 to the bath solution. If was elicited by using 1-s voltage steps from –130 to –50 mV applied at 5-s intervals. If was measured as the difference between the instantaneous inward current and that at the end of each voltage step.
Action potentials. In separate experiments, we used current-clamp recordings to evaluate resting potential and abnormal automaticity. For these recordings, the bath solution contained (in mM) 134 NaCl, 1 MgCl2, 5 KCl, 5 HEPES, 1.8 CaCl2, and 5 D-glucose; pH was adjusted to 7.40 with NaOH. Cells were stimulated at 0.2, 0.5, 1, and/or 2 Hz. The proportion of myocytes exhibiting abnormal automaticity was calculated. Zatebradine (10 µM) was used as an If blocker (12, 39).
Reagents. Zatebradine was supplied by Boehringer Ingleheim Pharma. All other reagents were purchased from Sigma Chemical (St. Louis, MO).
Data analysis. Cardiac hypertrophy was assessed by the heart weight-to-body weight ratio [heart wt (in mg)/body wt (in g)] (37). The measured currents were expressed as current density (in pA/pF) after normalization for cell capacitance. IK1 inward conductance (in mS/cm2) was determined by calculating the slope of the linear portion of the current density-voltage relationship from –140 mV to –100 mV (3). Peak outward IK1 density was measured at –60 mV (I–60). The IK1 rectification ratio was calculated as {[(I–100) – (I–60)]/I–100} x 100 (2, 24).
In pilot experiments, we constructed a threshold (>–0.60 pA/pF) for identification of If by identifying a value that included 95% of the end-stage heart failure myocytes while excluding 90% of the 2-mo-old control myocytes. To assess the physiological relevance of If, only cells classified as having significant If were included in the analysis of current differences at –90 mV.
If current density (in pA) was normalized to the cell capacitance (in pF) and expressed as pA/pF. All data were included for the current-voltage and current density-voltage relationship analyses.
Initial statistical analysis revealed no sex-dependent statistical differences; male and female data were pooled for further analyses. Two-way ANOVA with post hoc Student-Newman-Keuls test was used to test for differences between groups. One-way ANOVA was used to test for age-dependent cardiac hypertrophy in the SHHF rats. Differences in the proportion of cells demonstrating If (as defined above) were assessed by using the Pearson 
2-square test. Data are presented as means ± SE. Statistical significance was defined as P < 0.05. Statistical analysis was performed using SAS for Windows (version 8.0.1; SAS Systems, Cary, NC).
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RESULTS |
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17 mo (10.6 ± 1.6 vs. 3.2 ± 0.2 in SHHF rats and controls, respectively, P < 0.02). In controls, significant myocyte hypertrophy only occurred in the oldest group (Fig. 1).
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If.
Figure 3 depicts barium-insensitive current recordings (A and B) and summary data for ventricular If in Wistar-Furth and SHHF rats in all myocytes tested (C and D). There was an age-dependent increase in If in Wistar-Furth rats. In SHHF rats, the If was larger at all ages compared with 2-mo-old controls. There were no age- or heart failure-dependent increases in If density in the SHHF groups. When only cells expressing significant If (as defined in METHODS) were analyzed (Fig. 3, E and F), there were significantly more myocytes expressing If in the SHHF groups at both 2 and 8 mo of age. However, at 17 mo of age, there was no difference between control and SHHF rats in the proportion of myocytes expressing If. The amplitude of If increases as a function of myocyte size (Fig. 3F, P < 0.035).
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17 mo of age, there was no significant difference between groups in the If at –90 mV. We recorded action potentials from 2-mo-old (10 cells) and 8-mo-old (3 cells) SHHF rats. Action potentials were recorded in 11 myocytes from 2- to 6-mo-old Wistar rats; no age-dependent differences in action potential characteristics were found in the Wistar myocytes, and these data were pooled (resting membrane potential of –81.2 ± 2.6 and –82.4 ± 1.7 mV at 2 and 6 mo, respectively). There was no difference between 2-mo-old SHHF myocytes and control myocytes in the resting membrane potential. The resting membrane potential was reduced in the 8-mo-old SHHF myocytes compared with Wistar controls (P < 0.05). There was an age-dependent reduction in resting membrane potential in myocytes from the SHHF rats, because the 8-mo-old SHHF myocytes (where IK1 was reduced) were depolarized compared with 2-mo-old SHHF myocytes (–72.2 ± 1.8 vs. –80.6 ± 1.2 mV, respectively; P < 0.05).
In control myocytes, we did not observe any evidence of abnormal automaticity (0 of 11). In contrast, in the 2-mo-old SHHF myocytes, we observed abnormal automaticity in three of five myocytes at 0.2 Hz. At a faster stimulation rate of 0.5 Hz, none of the myocytes (zero of 8) from the 2-mo-old SHHF rats exhibited abnormal automaticity; in contrast, two out of three myocytes from the 8-mo-old SHHF rats exhibited abnormal automaticity at 0.5 Hz. Abnormal automaticity was abolished by zatebradine (If channel blocker) and reappeared after washout of zatebradine (Fig. 4). At stimulation rates of 1 and 2 Hz, no abnormal automaticity (0/13) was evident in the SHHF myocytes.
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DISCUSSION |
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80% of patients (11), and many with heart failure die of sudden cardiac death. We used the SHHF rat model and age-matched controls to evaluate the ventricular diastolic membrane currents, IK1 and If, and found differential regulation of these currents by hypertension, heart failure, and aging. SHHF rats are a reliable model for human hypertension and heart failure. Systolic blood pressures are consistently elevated in the SHHF rats but are decreased after the progression to significant heart failure (which typically occurs in phenotypically lean SHHF rats at 15–30 mo of age) (15, 26). Myocyte size is increased as early as 2 mo of age in SHHF rats, and progressive myocyte and cardiac hypertrophy have been reported through 12 mo of age in the SHHF rats (28). During heart failure, the SHHF rats have intracardiac thrombi, myocyte hypertrophy, and increased cardiac interstitial fibrosis (17).
Wistar and Wistar-Furth rats have been used as a suitable control for SHHF rats (8). Wistar-Furth rats have normal blood pressures during early to middle adult ages (14), although, recently, the systolic blood pressure in aged Wistar-Furth rats (21 mo old) was reported to be elevated, 160 mmHg (38). Thus it is possible that hypertension may have contributed to the increased myocyte capacitance and increased If in the aged controls.
We observed an age-dependent decrement in IK1 in the control group. This is in contrast to previous reports where no change in IK1 was reported when comparing ventricular myocytes from 3- and 9-mo-old (20) or young and senescent (24- to 25-mo-old) Wistar rats (40). Although we did observe a reduction in the aged control rats, there was a significantly greater reduction in IK1 with age and disease progression in SHHF rats. When compared with SHHF rats, the age-matched Wistar-Furth rats expressed a statistically decreased inward conductance only in the 17-mo age group. To our knowledge, this decrease in inward IK1 during aging has not been previously reported and could contribute to a depolarized diastolic membrane potential.
The peak outward IK1 was significantly altered only in end-stage SHHF rats, consistent with a heart failure-induced response. This reduction would be expected to result in a prolongation of terminal repolarization and has been previously described in other heart failure models (16, 32). We observed a significant reduction in the IK1 rectification ratio with age in both controls and SHHF rats. The rectification ratio decrease was more pronounced in SHHF rats than in Wistar-Furth rats. Rectification of IK1 is modulated by testosterone, intracellular magnesium, and intracellular polyamines; (2, 23, 27) alterations in these regulators have been reported during aging, hypertension and heart failure, (13, 19, 29, 33) and may be the basis of this observation.
In normal human and rat hearts, there is a low level of ventricular If, and the proportion of cells with significant If is less in controls, when compared with myocytes from failing hearts (5, 7). Ventricular If is increased in both amplitude and prevalence with both normal aging and hypertension in rats (4, 5). The age-dependent increase we observed in the proportion of myocytes expressing If in controls is similar to that reported in normal Wistar-Kyoto rats (5). Notably, the age dependence of the proportion of myocytes we observed with If in the SHHF rats differs from that previously reported in SHRs, where the occurrence of If in young SHRs did not differ from that in young controls (5). The reason for this difference is unclear; it is interesting to note that, whereas both SHRs and SHHF rats are hypertensive at a young age, only SHHF rats consistently progress to develop heart failure at an early age.
Similar to the observed increases in If in both control and SHHF rats, ventricular If is increased in human myocytes as a result of normal aging, hypertension, and heart failure (6, 7). We found that the amplitude of If was positively correlated with cell size, and this suggests that hypertrophy, and not heart failure per se, increases If, consistent with a previous report in hypertensive rats (4). This relationship explains why we did not observe a further increase in current density with progressive heart failure in the SHHF rats, as there was no further increase in myocyte capacitance (Fig. 1).
The early occurrence of If in the SHHF rats is consistent with the known response to hypertension (5). Thus our data suggest that hypertension can mediate increased If even at an early age in the SHHF rat. Progression from hypertension to hypertensive heart failure increases If amplitude, but not current density, because amplitude is tightly correlated with myocyte size. This differs from a previous report by Cerbai et al. (5) in SHRs where cell size did not correlate with If amplitude, although they did observe a significant correlation in controls.
The physiological relevance of the If expression in the SHHF rats can be evaluated by comparing the current at –90 mV (to approximate a normal resting potential). This reveals a significant increase in current amplitude as a function of age in both control and SHHF rats, although the current was always larger in the SHHF rats.
If is a time-dependent current, so we used variable action potential stimulation rates to assess the physiological impact of If. Our data indicate that increased If alone (as observed in the 2-mo-old SHHF rats) is sufficient to result in abnormal automaticity, only at very low stimulation rates (0.2 Hz). On the basis of the data from both 2- and 8-mo-old SHHF myocytes, both an increase in If and a reduction in inward IK1 are necessary for abnormal automaticity to occur at a faster stimulation rate (0.5 vs. 0.2 Hz). Our data suggest that altered excitability through a combination of decreases in IK1 and increases in If could precipitate arrhythmias during aging, hypertension, and heart failure.
Limitations. We did not directly measure blood pressure in our cohort of animals. However, the SHHF rats have reproducible blood pressures at the time points used in this study, which we obtained from the literature. The SHHF rats in this study were phenotypically lean, a mixture of animals heterozygous for or lacking the cp (corpulent) gene. This factor, and the use of both males and females, resulted in a more variable onset of heart failure than would be seen in genotypically lean rats of one sex. To control for this variable, we used echocardiography to define the presence of heart failure, using previously established criteria (8, 28).
In conclusion, by using young adult and aged SHHF rats and control rats, we found that aging, hypertension, and heart failure have variable effects on diastolic membrane currents. Reductions in IK1 and increases in If are both arrhythmogenic, and the magnitude of these changes is affected by age, hypertension, and heart failure. The finding of reduced IK1 in aged controls is interesting, as there are increased ventricular arrhythmias during normal aging in humans (10, 18). Our results indicate that the underlying cause of arrhythmias may vary during the progression from hypertension to heart failure. IK1 is functionally regulated by testosterone, intracellular magnesium, and intracellular polyamines, whereas If is regulated by the autonomic nervous system. This suggests that therapeutic interventions targeting each of these ionic currents may be required to minimize ventricular arrhythmias during the progression of hypertensive heart failure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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P < 0.05 compared with 8-mo-old WF rats; 
P < 0.05 compared with 2-mo-old SHHF rats; 
P < 0.05 compared with 
, 2-mo-old WF rats; 
, 8-mo-old WF rats; 
, 17-mo-old WF rats; 
, 2-mo-old SHHF rats; 
, 8-mo-old SHHF rats; 
, >17-mo-old SHHF rats). D: normalized inward IK1 conductance for WF (open bars) and SHHF (solid bars) rats at different ages. Numbers on each bar indicate total cells in each group. E: peak outward current at –60 mV as a function of strain at different ages. F: rectification ratio for WF and SHHF rats (*age-dependent change from 2-mo-old WF rats; 
