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Altered diaphragm muscle action potentials in zucker diabetic fatty (ZDF) rats
Respiratory Physiology & Neurobiology 153 (2006) 157–165
Altered diaphragm muscle action potentials in zucker diabetic fatty (ZDF) rats Erik van Lunteren ∗ , Michelle Moyer Department of Medicine (Pulmonary and Critical Care Division), Cleveland VA Medical Center and Case Western Reserve University, 10701 East Boulevard, Cleveland, OH 44106, USA Accepted 1 September 2005
Abstract The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes, being characterized by obesity, diabetes, and dyslipidemia. In vitro studies tested the hypothesis that diaphragm muscle from ZDF rats has abnormal resting membrane potential and action potentials, similar to type 1 diabetic rodents. Resting membrane potential was comparable for muscle from ZDF and control rats. Diaphragm from ZDF rats had augmented action potential peak height (92.1 mV versus 82.4 mV, P < 0.00001), overshoot (15.6 mV versus 8.1 mV, P < 0.001) and area (80.7 mV ms versus 68.6 mV ms, P < 0.001) compared with that from controls. Action potential rate of depolarization and repolarization were not affected. The K+ blocker, 3,4-diaminopyridine, augmented action potential duration and area of muscle from ZDF and controls, but without significant differences between animal groups. These findings in ZDF rats contrast with type 1 diabetic rats, suggesting that isolated hyperglycemia differs from hyperglycemia combined with other metabolic perturbations with respect to diaphragm electrophysiological derangements. Published by Elsevier B.V. Keywords: Type 2 diabetes; Metabolic syndrome; Obesity; Skeletal muscle; Diaphragm; Resting membrane potential; Action potential; Electrophysiology
1. Introduction Diabetes affects skeletal muscles electrophysiologically in two major manners. The first is a depolarization of resting membrane potential, which has been found in many (albeit not all) skeletal muscles (Grossie, 1982; ∗ Corresponding author. Tel.: +1 216 791 3800x4616/4611; fax: +1 216 231 3420. E-mail address: [email protected] (E. van Lunteren).
1569-9048/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.resp.2005.09.015
McGuire and MacDermott, 1998, 1999; McGuire et al., 2001; Paulus and Grossie, 1983; Lin-Shiau et al., 1993). The second is an abnormality in action potential waveform, with several specific changes having been described, including a diminution of amplitude, reduced rate of depolarization, both hastened and slowed repolarization, and reduced action potential area (Grossie, 1982; van Lunteren and Moyer, 2003). Of note is that these changes have thus far only been elucidated in type 1 diabetes models, which are typi-
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cally produced by the injection of agents toxic to the pancreas in normal rodents. Human type 2 diabetes is more common than type 1 diabetes in much of the Western world, and is becoming increasingly prevalent in many countries due to factors such as reduced exercise and increased obesity. It is therefore important to also define the skeletal muscle electrophysiological changes in diabetes associated with obesity. Types 1 and 2 diabetes differ considerably in their pathogenesis and pathophysiology, yet share many of their long-term complications. Hence, it is not clear if the two variants of the disease affect muscle electrophysiologically in the same manner. Several rodent models of concomittant obesity and various severities of diabetes exist, among which Zucker Diabetic Fatty (ZDF) rats also demonstrate a number of other features seen in the metabolic syndrome (Clark et al., 1983; Peterson et al., 1990; Sparks et al., 1998). The hypothesis of the present study is that ZDF rats have altered diaphragm muscle resting membrane potential and action potential waveforms, similar to what has been described in type 1 diabetic animals.
2. Methods Studies were done using the ZDF model of rat diabetes-obesity. All studies were approved by the local animal care and use committee. Male ZDF/GmiCrlfa/fa rats and lean controls (+/?) were obtained from Charles River Laboratories (2003). Homozygous (fa/fa) males develop obesity, dyslipidemia, and hyperglycemia, with non-fasting glucose levels of ∼500 mg/dl or higher at an age of 4 months, whereas lean fa/+ and +/+ animals remain normoglycemic (Peterson et al., 1990; http://www.criver.com/ RM/rats/documents/RM ZDF datasheet.pdf, accessed 3/15/05). ZDF rats were fed Purina 5008 chow, as recommended by the vendor. When the rats reached an age of 4 months they were anesthetized with urethane (initial dose 1 g/kg i.p., with additional doses of 0.1–0.2 g/kg given intraperitoneally as needed). At that time the lean animals weighed 364 ± 3 g and the ZDF rats weighed 475 ± 52 g. The diaphragm and both phrenic nerves were removed surgically and placed in oxygenated Krebs solution composed of (in mM): NaCl 135, KCl 5, CaCl2 2.5, MgSO4 1, NaH2 PO4 1,
NaHCO3 15, glucose 11, with pH adjusted to 7.25– 7.35, bubbled continuously with 95% O2 , 5% CO2 . The diaphragm, left intact to the ribs and the central tendon, was divided into two large sections, each with a phrenic nerve attached. All chemicals were obtained from Sigma Chemical (St. Louis, MO). Each hemi-diaphragm was pinned in a Sylgard-lined 35 mm petri dish. The muscle was stretched out with sufficient tension to prevent any sagging of fibers in order to facilitate penetration by the microelectrodes, as done previously (van Lunteren and Moyer, 1998, 2003; van Lunteren et al., 2001). Oxygenated Krebs solution flowed into the dish and the overflow was evacuated by suction. The solution was not bubbled directly in the petri dish in order to minimize vibration during electrophysiological recording. This set-up has been verified to be well-oxygenated using a dissolved oxygen meter (World Precision Instruments, Sarasota, FL). Temperature was regulated at 37 ◦ C in the Petri dish with a Peltier device (Medical Systems, Greenvale, NY) and monitored with a thermistor. Intracellular membrane potentials and action potentials of muscle fibers were recorded using glass microelectrodes fabricated with a Flaming Brown micropipette puller (Sutter Instruments, Novato, CA) (resistance 5–15 M when filled with 3 M KCl). Microelectrodes were lowered slowly by a micropositioner (David Kopf Instruments, Tujunga, CA). Action potentials were evoked using a single supramaximal stimulus applied to the phrenic nerve (pulse width 0.2 ms) via a suction electrode (A-M Systems, Everett, WA). Potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), digitized, collected on-line (Axotape software, Axon Instruments, Foster City, CA), and stored on the hard drive of a computer for future analysis. Similar to a previous study in a type 1 diabetic model (van Lunteren and Moyer, 2003), we used a K+ channel blocker 3,4-diaminopyridine (DAP, 0.3 mM) to further elucidate whether the regulation of action potentials by K+ channels was altered in muscle from ZDF animals. Repetitive muscle contraction dislodged the electrode from the cell, so that it was not feasible to study the same muscle cell before and after the addition of DAP. Therefore, several action potentials were recorded from the same hemi-diaphragm initially and
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then 10 min after the addition of DAP, with care taken not to record from the same fiber twice. The DAP dose of 0.3 mM was chosen based on the results of a previous study which identified this dose as being the smallest amount that gave a near maximal force increase (van Lunteren and Moyer, 1996). Action potentials were analyzed using a combination of the manually-driven cursors provided with the Axotape software and the semi-automated analysis capabilities of the Strathclyde Electrophysiology Software Whole Cell Program (distributed by Dagan, Minneapolis, MN). Small segments of data containing the action potential were exported from Axotape to Whole Cell Program. Action potential properties were characterized as follows: peak height (difference between resting membrane potential and the peak positive voltage), overshoot (amount by which the voltage at the peak of the action potential exceeded 0 mV), 10–90% rise time (time required for the depolarization phase to go from 10 to 90% of action potential height), maximal rate of rise (most rapid rate of depolarization), area (the integral of membrane potential during the action potential measured relative to resting membrane potential), and 50% decay time (T 50%, time required for the action potential to repolarize 50% of the way back to resting membrane potential, taken from the time of the peak of the action potential). Sample sizes were 5–7 hemi-diaphragms for each experimental arm. There were 5 lean and 5 ZDF rats used in this study, of which muscle from 4 of each group were also studied following DAP. Recordings were made from 36 and 22 muscle fibers from lean and ZDF rats, respectively, before DAP, and from 21 and 17 fibers from lean and ZDF rats, respectively, after DAP. Due to the fact that measurements were made from individual muscle fibers rather than the muscle as a whole, statistical analysis was performed by using both the cell and the animal as an experimental unit. Results of both approaches are presented in the figure legends, and in all cases conclusions derived with the two methods were consistent with each other. All values presented are mean ± S.E. Statistical analysis of the action potential measurements in fibers from ZDF versus lean animals, and for before versus after DAP, was done with an unpaired t-test. For statistical analysis, a P value of <0.05 (two-tailed) was chosen as indicative of significance.
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3. Results Resting membrane potential did not differ between diaphragm muscle of lean and ZDF rats (−73 ± 1 and −75 ± 1 mV, respectively). Furthermore, the K+ channel blocker DAP did not affect resting membrane potential of either group (−72 ± 2 and −74 ± 2 mV after the addition of DAP for lean and ZDF rats, respectively). Examples of action potentials recorded from the diaphragm of lean and ZDF rats are depicted in Fig. 1. The peak height of the action potential in diaphragm from ZDF rats was significantly greater than that of the lean rats, as was the action potential overshoot (Figs. 1 and 2). The increase in peak amplitude of the action potential of the ZDF muscle resulted in a significant increase in action potential area compared to lean muscle. On the other hand, the depolarization phase of the action potential, as measured by rate of rise (in mV/ms) and rise time (in ms), and the rate of action potential repolarization, as measured by T 50%, were similar for muscles from lean and ZDF animals (Fig. 3). DAP did not alter action potential peak height or rate of depolarization for muscle fibers from either lean or ZDF rats (Fig. 4). In lean animals action potential area increased by 141% and repolarization time by 117% with the addition of DAP (Fig. 5, top). In ZDF animals, the DAP-induced increases were 117% for action potential area and 143% for repolarization time (Fig. 5, bottom). There was considerably more diversity among fibers in these two parameters after compared with before DAP. As a result, any tendencies towards differences between muscle from lean and
Fig. 1. Examples of diaphragm action potentials recorded from lean and ZDF rats. Traces are superimposed to facilitate comparison. The action potential from the ZDF rat muscle was characterized by higher peak, greater overshoot and larger area than that from the lean rat.
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Fig. 2. Values for action potential peak height, overshoot and area in lean and ZDF rats (mean ± S.E.). Asterisks (* ) denote significant differences between lean and ZDF data. Significantly larger values were noted in ZDF than lean rats for all three parameters, whether analyzed statistically on the basis of fibers (P < 0.00001, P < 0.001, and P < 0.001, respectively) or on the basis of animals (P < 0.0001, P < 0.05, and P = 0.05, respectively).
ZDF rats in the presence of DAP did not reach statistical significance (P > 0.40 for all instances, irrespective of whether fibers or animals were compared).
4. Discussion The Zucker rat is characterized by homozygosity for the fa gene, and is obese and hyperinsulinemic but generally euglycemic. The frankly hyperglycemic trait (initially called diabetic Zucker fatty, later named Zucker diabetic fatty or ZDF) originated in 1978 in an outbred line of Zucker rats (Clark et al., 1983), has been rederived on several occasions from groups of animals with diabetic lineage, and has been maintained as an inbred line subsequently (Peterson et al., 1990). Male obese ZDF rats (fa/fa), but not lean rats (fa/+ or +/+), develop diabetes. Hyperglycemia starts at an age of around 7 weeks and is fully established by 9–10 weeks of age. The animals also become progressively more dyslipidemic as they become older (Sparks et al.,
1998). Thus ZDF rats are an attractive animal model for human type 2 diabetes as well as the metabolic syndrome. A number of studies have examined skeletal muscle resting membrane potential in streptozotocin-induced or alloxan-induced diabetes (type 1 models). One group found no effects of diabetes on sternohyoid (McGuire et al., 2001) or diaphragm (McGuire and MacDermott, 1998) resting membrane potential, yet extensor digitorum longus and soleus muscle were significantly depolarized (McGuire and MacDermott, 1999). Others have found depolarization of extensor digitorum longus (Grossie, 1982; Paulus and Grossie, 1983) and diaphragm (Lin-Shiau et al., 1993) muscles by diabetes, with which our previous diaphragm studies are in agreement (van Lunteren and Moyer, 2003). Thus most evidence suggests that type 1 diabetes depolarizes muscle resting membrane potential, although with some heterogeneity in presence and extent of response. The extent of depolarization reported is in the range of 2–15 mV (Grossie, 1982; McGuire and MacDermott,
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Fig. 3. Values for action potential rate of rise, rise time and rate of repolarization in lean and ZDF rats (mean ± S.E.). No significant differences were noted between diaphragm fibers of the two rat types.
1999; Paulus and Grossie, 1983; Lin-Shiau et al., 1993; van Lunteren and Moyer, 2003). Altered resting potential has been associated with reduced resting K+ (Grossie, 1982) and Cl− (Lin-Shiau et al., 1993) conductances. In contrast, the present study found no changes in resting membrane potential in ZDF compared with lean animals, arguing against changes in muscle resting ionic conductances in the ZDF muscle. We are aware of only two studies examining effects of diabetes on skeletal muscle action potentials, again both conducted in a type 1 model. The first study (Grossie, 1982) examined limb muscle in alloxandiabetic animals of two severities, with one group having severe enough disease to result in weight loss and high mortality rates in the absence of exogenous insulin, and the other not requiring treatment for survival. Muscle from the more severe group had substantially reduced amplitude and rate of depolarization, and increased duration, of action potentials, and these abnormalities occurred within days after discontinuing insulin therapy. Muscle from animals with mild diabetes had more modest action potential changes,
which for the most part developed at a slower rate after cessation of insulin (over the course of 2–5 weeks). The second study (van Lunteren and Moyer, 2003) examined diaphragm muscle 8 weeks after streptozotocin injection in diabetic animals without weight loss. There was a hastening of action potential repolarization resulting in a reduced action potential area, but no changes in action potential height or rate of depolarization. We are not aware of any data examining either resting membrane potential or action potentials in skeletal muscle from either humans with, or animal models of, type 2 diabetes. The present study in the ZDF rat demonstrates changes not seen in either of the previous studies in the type 1 diabetic models, in that action potential height and overshoot were increased, and action potential area was augmented. These electrophysiological changes in ZDF muscle should, if anything, improve muscle force rather than reduce force, as the augmented action potential size would be expected to increase Ca2+ influx (Miledi et al., 1984). Direct studies of muscle contractility in the ZDF model are needed to determine whether this is indeed the case.
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Fig. 4. Effect of 3,4-diaminopyridine (DAP) on action potential peak height and rate of depolarization in muscle from lean and ZDF animals (mean ± S.E.). No significant changes were found in these parameters.
DAP and other members of the aminopyridine family (e.g. 4-aminopyridine) block many types of K+ channels, with some channels being blocked better than others but not to a sufficient extent that a high degree
of specificity among K+ channel types can be assigned to these agents. Voltage gated K+ channels of skeletal muscle are well-documented to be blocked by 4aminopyridine (Gillespie and Hutter, 1975; Gillespie,
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Fig. 5. Effect of 3,4-diaminopyridine (DAP) on action potential area and rate of repolarization (T 50%) in muscle from lean and ZDF animals (mean ± S.E.). Asterisks (* ) denote significant changes induced by DAP. In lean animals both parameters were increased significantly by DAP irrespective of whether data were analyzed on the basis of fibers (P < 0.0000001 and P < 0.0000001, respectively) or animals (P < 0.05 and P < 0.005, respectively), as depicted in the top panels. Similarly, DAP increased these parameters significantly in ZDF animals irrespective of whether data were analyzed on the basis of fibers (P < 0.00002 and P < 0.00002, respectively) or animals (P < 0.05 and P < 0.05, respectively), as depicted in the bottom panels.
1977). The aminopyridines slow action potential repolarization and increase action potential area to greater extents than do a number of other K+ channel blockers, such as tetraethylammonium, Ba2+ , glibenclamide, apamin and charybdotoxin (van Lunteren et al., 2001). In diaphragm muscle from type 1 diabetic animals, DAP increased action potential area and slowed the 50% repolarization time to a significantly smaller extent than seen in non-diabetic animals (van Lunteren and Moyer, 2003). In contrast, in the present study, differences in action potential area between ZDF and control animals were virtually identical in the presence of DAP compared to the absence of DAP (13 mV ms versus 14 mV ms, respectively). This difference was statistically significant for control animals but not ZDF animals, but this can be attributed to larger variability
among ZDF rats than among control animals, rather than differences in the magnitude of the effect. These data would therefore argue against a major role for DAP-sensitive K+ channels in action potential alterations found in ZDF compared to control rats. Furthermore, the role of DAP-sensitive K+ channels in producing action potential changes in diabetic versus non-diabetic animals was far more prominent in the type 1 diabetic model (van Lunteren and Moyer, 2003) than the currently studied type 2 diabetic model. Several factors may contribute to increases in action potential peak and overshoot: more vigorous increase in conductance of Na+ channels (either more channels that open, or channels staying open for a greater proportion of the time), a longer total duration of Na+ channel opening, delayed opening of K+ channels, or
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less vigorous increase in conductance of K+ channels. One would expect that a longer duration of Na+ channel opening and/or delayed onset of K+ channel opening should prolong the depolarization phase of the action potential, whereas lower K+ channel conductance would slow action potential repolarization, neither of which was found. It is possible that the increased peak/overshoot of ZDF compared with lean diaphragm is due to increased Na+ channel conductance, although there are no direct data available to determine whether this is indeed the case. Further studies are needed which address this possibility more directly. The present study focused on a single age of 4 months. As noted above, hyperglycemia in the ZDF model starts around week 7 and is fully established by week 9 or 10. Thus the animals in the present study most likely were hyperglycemic for approximately two months. This duration of hyperglycemia is therefore roughly comparable to that used in a previous study from our lab in type 1 diabetic animals (van Lunteren and Moyer, 2003), in which muscles were studied 8 weeks after the induction of diabetes with streptozotocin. Hence, duration of hyperglycemia is unlikely to explain differences between the present data and that from our previous study in the type 1 diabetic model. In conclusion, diaphragm muscle from 4-month old ZDF rats has altered action potentials compared with lean control animals, manifested by increased action potential height, overshoot and area. This contrasts with muscle action potential changes previously described in type 1 diabetic models. Furthermore ZDF animals had no alterations in skeletal muscle resting membrane potential, in contrast to the depolarization, which has been found in many studies of type 1 diabetic rodents. The ZDF rat is a complex model, with hyperglycemia, lipid dysregulation and obesity, and with both genetic and dietary factors contributing to the development and severity of disease. These alterations change the metabolic milieu in which the muscles function, as well as the mechanical load imposed on the muscles as a result of the increased body weight. Thus factors other than, or in combination with, the hyperglycemia could explain why ZDF rats and type 1 induced-diabetic rats differ with regards to skeletal muscle electrophysiological perturbations. Future studies are needed to examine the mechanical consequences of, and to better define the ionic conductance changes which underly, the action potential changes.
Acknowledgments This study was supported in part by a Merit Review Grant from the VA Research Service, as well as NIH Grant HL-70697. References Charles River Laboratories, 2003. ZDF Rat [Online]. Charles River Laboratories. http://www.criver.com/RM/rats/documents/ RM ZDF datasheet.pdf [accessed 15 March 2005]. Clark, J.B., Palmer, C.J., Shaw, W.N., 1983. The diabetic Zucker fatty rat. Proc. Soc. Exp. Biol. Med. 173, 68–75. Gillespie, J.I., 1977. Voltage-dependent blockage of the delayed potassium current in skeletal muscle by 4-aminopyridine. J. Physiol. (London) 273, 64–65. Gillespie, J.I., Hutter, O.F., 1975. The actions of 4-aminopyridine on the delayed potassium current in skeletal muscle fibers. J. Physiol. (London) 252, 70–71. Grossie, J., 1982. Contractile and electrical characteristics of extensor muscle from alloxan-diabetic rats. An in vitro study. Diabetes 31, 194–202. Lin-Shiau, S.Y., Liu, S.H., Lin, M.J., 1993. Use of ion channel blockers in the exploration of possible mechanisms involved in the myopathy of diabetic mice. Naunyn-Schmiedeberg’s Arch. Pharmacol. 348, 311–318. McGuire, M., MacDermott, M., 1998. The influence of streptozotocin-induced diabetes and the antihyperglycaemic agent metformin on the contractile characteristics and the membrane potential of the rat diaphragm. Exp. Physiol. 83, 481–487. McGuire, M., MacDermott, M., 1999. The influence of streptozotocin diabetes and metformin on erythrocyte volume and on the membrane potential and the contractile characteristics of the extensor digitorum longus and soleus muscles in rats. Exp. Physiol. 84, 1051–1058. McGuire, M., Dumbleton, M., MacDermott, M., Bradford, A., 2001. Contractile and electrical properties of sternohyoid muscle in streptozotocin diabetic rats. Clin. Exp. Pharmacol. Physiol. 28, 184–187. Miledi, R., Parker, I., Zhu, P.H., 1984. Extracellular ions and excitation-contraction coupling in frog twitch muscle fibers. J. Physiol. (London) 351, 687–710. Paulus, S.F., Grossie, J., 1983. Skeletal muscle in alloxan diabetes: a comparison of isometric contractions in fast and slow muscle. Diabetes 32, 1035–1039. Peterson, R.G., Shaw, W.N., Neel, M.-A., Little, L., Eichberg, J., 1990. Zucker diabetic fatty rat as a model for non-insulin dependent diabetes mellitus. Inst. Lab. Anim. Resources News 32, 16–27. Sparks, J.D., Phung, T.L., Bolognino, M., Cianci, J., Khurana, R., Peterson, R.G., Sowden, M.P., Corsetti, J.P., Sparks, C.E., 1998. Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinoponic hyperglycemia. Metabolism 47, 1315–1324.
E. van Lunteren, M. Moyer / Respiratory Physiology & Neurobiology 153 (2006) 157–165 van Lunteren, E., Moyer, M., 1996. Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure. J. Appl. Physiol. 81, 2214–2220. van Lunteren, E., Moyer, M., 1998. Electrophysiologic and inotropic effects of K+ -channel blockade in aged diaphragm. Am. J. Respir. Crit. Care Med. 158, 820–826.
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van Lunteren, E., Moyer, M., Dick, T.E., 2001. Modulation of diaphragm action potentials by K+ channel blockers. Respir. Physiol. 124, 217–230. van Lunteren, E., Moyer, M., 2003. Streptozotocin-diabetes alters action potentials in rat diaphragm. Resp. Physiol. Neurobiol. 135, 9–16.
Altered diaphragm muscle action potentials in zucker diabetic fatty (ZDF) rats Erik van Lunteren ∗ , Michelle Moyer Department of Medicine (Pulmonary and Critical Care Division), Cleveland VA Medical Center and Case Western Reserve University, 10701 East Boulevard, Cleveland, OH 44106, USA Accepted 1 September 2005
Abstract The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes, being characterized by obesity, diabetes, and dyslipidemia. In vitro studies tested the hypothesis that diaphragm muscle from ZDF rats has abnormal resting membrane potential and action potentials, similar to type 1 diabetic rodents. Resting membrane potential was comparable for muscle from ZDF and control rats. Diaphragm from ZDF rats had augmented action potential peak height (92.1 mV versus 82.4 mV, P < 0.00001), overshoot (15.6 mV versus 8.1 mV, P < 0.001) and area (80.7 mV ms versus 68.6 mV ms, P < 0.001) compared with that from controls. Action potential rate of depolarization and repolarization were not affected. The K+ blocker, 3,4-diaminopyridine, augmented action potential duration and area of muscle from ZDF and controls, but without significant differences between animal groups. These findings in ZDF rats contrast with type 1 diabetic rats, suggesting that isolated hyperglycemia differs from hyperglycemia combined with other metabolic perturbations with respect to diaphragm electrophysiological derangements. Published by Elsevier B.V. Keywords: Type 2 diabetes; Metabolic syndrome; Obesity; Skeletal muscle; Diaphragm; Resting membrane potential; Action potential; Electrophysiology
1. Introduction Diabetes affects skeletal muscles electrophysiologically in two major manners. The first is a depolarization of resting membrane potential, which has been found in many (albeit not all) skeletal muscles (Grossie, 1982; ∗ Corresponding author. Tel.: +1 216 791 3800x4616/4611; fax: +1 216 231 3420. E-mail address: [email protected] (E. van Lunteren).
1569-9048/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.resp.2005.09.015
McGuire and MacDermott, 1998, 1999; McGuire et al., 2001; Paulus and Grossie, 1983; Lin-Shiau et al., 1993). The second is an abnormality in action potential waveform, with several specific changes having been described, including a diminution of amplitude, reduced rate of depolarization, both hastened and slowed repolarization, and reduced action potential area (Grossie, 1982; van Lunteren and Moyer, 2003). Of note is that these changes have thus far only been elucidated in type 1 diabetes models, which are typi-
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cally produced by the injection of agents toxic to the pancreas in normal rodents. Human type 2 diabetes is more common than type 1 diabetes in much of the Western world, and is becoming increasingly prevalent in many countries due to factors such as reduced exercise and increased obesity. It is therefore important to also define the skeletal muscle electrophysiological changes in diabetes associated with obesity. Types 1 and 2 diabetes differ considerably in their pathogenesis and pathophysiology, yet share many of their long-term complications. Hence, it is not clear if the two variants of the disease affect muscle electrophysiologically in the same manner. Several rodent models of concomittant obesity and various severities of diabetes exist, among which Zucker Diabetic Fatty (ZDF) rats also demonstrate a number of other features seen in the metabolic syndrome (Clark et al., 1983; Peterson et al., 1990; Sparks et al., 1998). The hypothesis of the present study is that ZDF rats have altered diaphragm muscle resting membrane potential and action potential waveforms, similar to what has been described in type 1 diabetic animals.
2. Methods Studies were done using the ZDF model of rat diabetes-obesity. All studies were approved by the local animal care and use committee. Male ZDF/GmiCrlfa/fa rats and lean controls (+/?) were obtained from Charles River Laboratories (2003). Homozygous (fa/fa) males develop obesity, dyslipidemia, and hyperglycemia, with non-fasting glucose levels of ∼500 mg/dl or higher at an age of 4 months, whereas lean fa/+ and +/+ animals remain normoglycemic (Peterson et al., 1990; http://www.criver.com/ RM/rats/documents/RM ZDF datasheet.pdf, accessed 3/15/05). ZDF rats were fed Purina 5008 chow, as recommended by the vendor. When the rats reached an age of 4 months they were anesthetized with urethane (initial dose 1 g/kg i.p., with additional doses of 0.1–0.2 g/kg given intraperitoneally as needed). At that time the lean animals weighed 364 ± 3 g and the ZDF rats weighed 475 ± 52 g. The diaphragm and both phrenic nerves were removed surgically and placed in oxygenated Krebs solution composed of (in mM): NaCl 135, KCl 5, CaCl2 2.5, MgSO4 1, NaH2 PO4 1,
NaHCO3 15, glucose 11, with pH adjusted to 7.25– 7.35, bubbled continuously with 95% O2 , 5% CO2 . The diaphragm, left intact to the ribs and the central tendon, was divided into two large sections, each with a phrenic nerve attached. All chemicals were obtained from Sigma Chemical (St. Louis, MO). Each hemi-diaphragm was pinned in a Sylgard-lined 35 mm petri dish. The muscle was stretched out with sufficient tension to prevent any sagging of fibers in order to facilitate penetration by the microelectrodes, as done previously (van Lunteren and Moyer, 1998, 2003; van Lunteren et al., 2001). Oxygenated Krebs solution flowed into the dish and the overflow was evacuated by suction. The solution was not bubbled directly in the petri dish in order to minimize vibration during electrophysiological recording. This set-up has been verified to be well-oxygenated using a dissolved oxygen meter (World Precision Instruments, Sarasota, FL). Temperature was regulated at 37 ◦ C in the Petri dish with a Peltier device (Medical Systems, Greenvale, NY) and monitored with a thermistor. Intracellular membrane potentials and action potentials of muscle fibers were recorded using glass microelectrodes fabricated with a Flaming Brown micropipette puller (Sutter Instruments, Novato, CA) (resistance 5–15 M when filled with 3 M KCl). Microelectrodes were lowered slowly by a micropositioner (David Kopf Instruments, Tujunga, CA). Action potentials were evoked using a single supramaximal stimulus applied to the phrenic nerve (pulse width 0.2 ms) via a suction electrode (A-M Systems, Everett, WA). Potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), digitized, collected on-line (Axotape software, Axon Instruments, Foster City, CA), and stored on the hard drive of a computer for future analysis. Similar to a previous study in a type 1 diabetic model (van Lunteren and Moyer, 2003), we used a K+ channel blocker 3,4-diaminopyridine (DAP, 0.3 mM) to further elucidate whether the regulation of action potentials by K+ channels was altered in muscle from ZDF animals. Repetitive muscle contraction dislodged the electrode from the cell, so that it was not feasible to study the same muscle cell before and after the addition of DAP. Therefore, several action potentials were recorded from the same hemi-diaphragm initially and
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then 10 min after the addition of DAP, with care taken not to record from the same fiber twice. The DAP dose of 0.3 mM was chosen based on the results of a previous study which identified this dose as being the smallest amount that gave a near maximal force increase (van Lunteren and Moyer, 1996). Action potentials were analyzed using a combination of the manually-driven cursors provided with the Axotape software and the semi-automated analysis capabilities of the Strathclyde Electrophysiology Software Whole Cell Program (distributed by Dagan, Minneapolis, MN). Small segments of data containing the action potential were exported from Axotape to Whole Cell Program. Action potential properties were characterized as follows: peak height (difference between resting membrane potential and the peak positive voltage), overshoot (amount by which the voltage at the peak of the action potential exceeded 0 mV), 10–90% rise time (time required for the depolarization phase to go from 10 to 90% of action potential height), maximal rate of rise (most rapid rate of depolarization), area (the integral of membrane potential during the action potential measured relative to resting membrane potential), and 50% decay time (T 50%, time required for the action potential to repolarize 50% of the way back to resting membrane potential, taken from the time of the peak of the action potential). Sample sizes were 5–7 hemi-diaphragms for each experimental arm. There were 5 lean and 5 ZDF rats used in this study, of which muscle from 4 of each group were also studied following DAP. Recordings were made from 36 and 22 muscle fibers from lean and ZDF rats, respectively, before DAP, and from 21 and 17 fibers from lean and ZDF rats, respectively, after DAP. Due to the fact that measurements were made from individual muscle fibers rather than the muscle as a whole, statistical analysis was performed by using both the cell and the animal as an experimental unit. Results of both approaches are presented in the figure legends, and in all cases conclusions derived with the two methods were consistent with each other. All values presented are mean ± S.E. Statistical analysis of the action potential measurements in fibers from ZDF versus lean animals, and for before versus after DAP, was done with an unpaired t-test. For statistical analysis, a P value of <0.05 (two-tailed) was chosen as indicative of significance.
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3. Results Resting membrane potential did not differ between diaphragm muscle of lean and ZDF rats (−73 ± 1 and −75 ± 1 mV, respectively). Furthermore, the K+ channel blocker DAP did not affect resting membrane potential of either group (−72 ± 2 and −74 ± 2 mV after the addition of DAP for lean and ZDF rats, respectively). Examples of action potentials recorded from the diaphragm of lean and ZDF rats are depicted in Fig. 1. The peak height of the action potential in diaphragm from ZDF rats was significantly greater than that of the lean rats, as was the action potential overshoot (Figs. 1 and 2). The increase in peak amplitude of the action potential of the ZDF muscle resulted in a significant increase in action potential area compared to lean muscle. On the other hand, the depolarization phase of the action potential, as measured by rate of rise (in mV/ms) and rise time (in ms), and the rate of action potential repolarization, as measured by T 50%, were similar for muscles from lean and ZDF animals (Fig. 3). DAP did not alter action potential peak height or rate of depolarization for muscle fibers from either lean or ZDF rats (Fig. 4). In lean animals action potential area increased by 141% and repolarization time by 117% with the addition of DAP (Fig. 5, top). In ZDF animals, the DAP-induced increases were 117% for action potential area and 143% for repolarization time (Fig. 5, bottom). There was considerably more diversity among fibers in these two parameters after compared with before DAP. As a result, any tendencies towards differences between muscle from lean and
Fig. 1. Examples of diaphragm action potentials recorded from lean and ZDF rats. Traces are superimposed to facilitate comparison. The action potential from the ZDF rat muscle was characterized by higher peak, greater overshoot and larger area than that from the lean rat.
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Fig. 2. Values for action potential peak height, overshoot and area in lean and ZDF rats (mean ± S.E.). Asterisks (* ) denote significant differences between lean and ZDF data. Significantly larger values were noted in ZDF than lean rats for all three parameters, whether analyzed statistically on the basis of fibers (P < 0.00001, P < 0.001, and P < 0.001, respectively) or on the basis of animals (P < 0.0001, P < 0.05, and P = 0.05, respectively).
ZDF rats in the presence of DAP did not reach statistical significance (P > 0.40 for all instances, irrespective of whether fibers or animals were compared).
4. Discussion The Zucker rat is characterized by homozygosity for the fa gene, and is obese and hyperinsulinemic but generally euglycemic. The frankly hyperglycemic trait (initially called diabetic Zucker fatty, later named Zucker diabetic fatty or ZDF) originated in 1978 in an outbred line of Zucker rats (Clark et al., 1983), has been rederived on several occasions from groups of animals with diabetic lineage, and has been maintained as an inbred line subsequently (Peterson et al., 1990). Male obese ZDF rats (fa/fa), but not lean rats (fa/+ or +/+), develop diabetes. Hyperglycemia starts at an age of around 7 weeks and is fully established by 9–10 weeks of age. The animals also become progressively more dyslipidemic as they become older (Sparks et al.,
1998). Thus ZDF rats are an attractive animal model for human type 2 diabetes as well as the metabolic syndrome. A number of studies have examined skeletal muscle resting membrane potential in streptozotocin-induced or alloxan-induced diabetes (type 1 models). One group found no effects of diabetes on sternohyoid (McGuire et al., 2001) or diaphragm (McGuire and MacDermott, 1998) resting membrane potential, yet extensor digitorum longus and soleus muscle were significantly depolarized (McGuire and MacDermott, 1999). Others have found depolarization of extensor digitorum longus (Grossie, 1982; Paulus and Grossie, 1983) and diaphragm (Lin-Shiau et al., 1993) muscles by diabetes, with which our previous diaphragm studies are in agreement (van Lunteren and Moyer, 2003). Thus most evidence suggests that type 1 diabetes depolarizes muscle resting membrane potential, although with some heterogeneity in presence and extent of response. The extent of depolarization reported is in the range of 2–15 mV (Grossie, 1982; McGuire and MacDermott,
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Fig. 3. Values for action potential rate of rise, rise time and rate of repolarization in lean and ZDF rats (mean ± S.E.). No significant differences were noted between diaphragm fibers of the two rat types.
1999; Paulus and Grossie, 1983; Lin-Shiau et al., 1993; van Lunteren and Moyer, 2003). Altered resting potential has been associated with reduced resting K+ (Grossie, 1982) and Cl− (Lin-Shiau et al., 1993) conductances. In contrast, the present study found no changes in resting membrane potential in ZDF compared with lean animals, arguing against changes in muscle resting ionic conductances in the ZDF muscle. We are aware of only two studies examining effects of diabetes on skeletal muscle action potentials, again both conducted in a type 1 model. The first study (Grossie, 1982) examined limb muscle in alloxandiabetic animals of two severities, with one group having severe enough disease to result in weight loss and high mortality rates in the absence of exogenous insulin, and the other not requiring treatment for survival. Muscle from the more severe group had substantially reduced amplitude and rate of depolarization, and increased duration, of action potentials, and these abnormalities occurred within days after discontinuing insulin therapy. Muscle from animals with mild diabetes had more modest action potential changes,
which for the most part developed at a slower rate after cessation of insulin (over the course of 2–5 weeks). The second study (van Lunteren and Moyer, 2003) examined diaphragm muscle 8 weeks after streptozotocin injection in diabetic animals without weight loss. There was a hastening of action potential repolarization resulting in a reduced action potential area, but no changes in action potential height or rate of depolarization. We are not aware of any data examining either resting membrane potential or action potentials in skeletal muscle from either humans with, or animal models of, type 2 diabetes. The present study in the ZDF rat demonstrates changes not seen in either of the previous studies in the type 1 diabetic models, in that action potential height and overshoot were increased, and action potential area was augmented. These electrophysiological changes in ZDF muscle should, if anything, improve muscle force rather than reduce force, as the augmented action potential size would be expected to increase Ca2+ influx (Miledi et al., 1984). Direct studies of muscle contractility in the ZDF model are needed to determine whether this is indeed the case.
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Fig. 4. Effect of 3,4-diaminopyridine (DAP) on action potential peak height and rate of depolarization in muscle from lean and ZDF animals (mean ± S.E.). No significant changes were found in these parameters.
DAP and other members of the aminopyridine family (e.g. 4-aminopyridine) block many types of K+ channels, with some channels being blocked better than others but not to a sufficient extent that a high degree
of specificity among K+ channel types can be assigned to these agents. Voltage gated K+ channels of skeletal muscle are well-documented to be blocked by 4aminopyridine (Gillespie and Hutter, 1975; Gillespie,
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Fig. 5. Effect of 3,4-diaminopyridine (DAP) on action potential area and rate of repolarization (T 50%) in muscle from lean and ZDF animals (mean ± S.E.). Asterisks (* ) denote significant changes induced by DAP. In lean animals both parameters were increased significantly by DAP irrespective of whether data were analyzed on the basis of fibers (P < 0.0000001 and P < 0.0000001, respectively) or animals (P < 0.05 and P < 0.005, respectively), as depicted in the top panels. Similarly, DAP increased these parameters significantly in ZDF animals irrespective of whether data were analyzed on the basis of fibers (P < 0.00002 and P < 0.00002, respectively) or animals (P < 0.05 and P < 0.05, respectively), as depicted in the bottom panels.
1977). The aminopyridines slow action potential repolarization and increase action potential area to greater extents than do a number of other K+ channel blockers, such as tetraethylammonium, Ba2+ , glibenclamide, apamin and charybdotoxin (van Lunteren et al., 2001). In diaphragm muscle from type 1 diabetic animals, DAP increased action potential area and slowed the 50% repolarization time to a significantly smaller extent than seen in non-diabetic animals (van Lunteren and Moyer, 2003). In contrast, in the present study, differences in action potential area between ZDF and control animals were virtually identical in the presence of DAP compared to the absence of DAP (13 mV ms versus 14 mV ms, respectively). This difference was statistically significant for control animals but not ZDF animals, but this can be attributed to larger variability
among ZDF rats than among control animals, rather than differences in the magnitude of the effect. These data would therefore argue against a major role for DAP-sensitive K+ channels in action potential alterations found in ZDF compared to control rats. Furthermore, the role of DAP-sensitive K+ channels in producing action potential changes in diabetic versus non-diabetic animals was far more prominent in the type 1 diabetic model (van Lunteren and Moyer, 2003) than the currently studied type 2 diabetic model. Several factors may contribute to increases in action potential peak and overshoot: more vigorous increase in conductance of Na+ channels (either more channels that open, or channels staying open for a greater proportion of the time), a longer total duration of Na+ channel opening, delayed opening of K+ channels, or
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less vigorous increase in conductance of K+ channels. One would expect that a longer duration of Na+ channel opening and/or delayed onset of K+ channel opening should prolong the depolarization phase of the action potential, whereas lower K+ channel conductance would slow action potential repolarization, neither of which was found. It is possible that the increased peak/overshoot of ZDF compared with lean diaphragm is due to increased Na+ channel conductance, although there are no direct data available to determine whether this is indeed the case. Further studies are needed which address this possibility more directly. The present study focused on a single age of 4 months. As noted above, hyperglycemia in the ZDF model starts around week 7 and is fully established by week 9 or 10. Thus the animals in the present study most likely were hyperglycemic for approximately two months. This duration of hyperglycemia is therefore roughly comparable to that used in a previous study from our lab in type 1 diabetic animals (van Lunteren and Moyer, 2003), in which muscles were studied 8 weeks after the induction of diabetes with streptozotocin. Hence, duration of hyperglycemia is unlikely to explain differences between the present data and that from our previous study in the type 1 diabetic model. In conclusion, diaphragm muscle from 4-month old ZDF rats has altered action potentials compared with lean control animals, manifested by increased action potential height, overshoot and area. This contrasts with muscle action potential changes previously described in type 1 diabetic models. Furthermore ZDF animals had no alterations in skeletal muscle resting membrane potential, in contrast to the depolarization, which has been found in many studies of type 1 diabetic rodents. The ZDF rat is a complex model, with hyperglycemia, lipid dysregulation and obesity, and with both genetic and dietary factors contributing to the development and severity of disease. These alterations change the metabolic milieu in which the muscles function, as well as the mechanical load imposed on the muscles as a result of the increased body weight. Thus factors other than, or in combination with, the hyperglycemia could explain why ZDF rats and type 1 induced-diabetic rats differ with regards to skeletal muscle electrophysiological perturbations. Future studies are needed to examine the mechanical consequences of, and to better define the ionic conductance changes which underly, the action potential changes.
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