Effects of streptozotocin-induced diabetes on action potentials in the sinoatrial node compared with other regions of the rat heart

In vivo biotelemetry studies have demonstrated that heart rate (HR) is progressively and rapidly reduced after administration of streptozotocin (STZ) and that the reduction in HR can be partially normalized with insulin replacement. Reductions in HR have also been reported in isolated perfused heart and superfused right atrial preparations suggesting that intrinsic defects in the heart are at least partly responsible for the bradycardia. The regional effects of STZ-induced diabetes mellitus (DM) on action potentials (APs) in the sinoatrial node (SAN), right and left atria and ventricles have been compared in the spontaneously beating Langendorff perfused rat heart 10–12 weeks after treatment. HR was significantly reduced in STZ-induced diabetic rat heart (174 ± 9 BPM) compared to controls (241 ± 12 BPM). The duration of AP repolarization at 50% and 70% from peak AP was significantly prolonged in SAN, right atrium and right ventricle from STZ-induced diabetic rat compared to age-matched controls. In the SAN AP duration (APD) at 50% and 70% were 51.7 ± 2.2 and 59.5 ± 2.3 ms in diabetic rat heart compared to 45.2 ± 1.7 and 50.0 ± 1.6 ms in controls, respectively. In contrast APD at 50% and 70% were not significantly altered in the left atrium and left ventricle. Regional defects in the expression and/or electrophysiology of SAN ion channels, and in particular those involved in AP repolarization, might underlie heart rhythm disturbances in the STZ-induced DM rat.     

Opposite effects of adrenaline and ouabain on the resting potential of rat atrial cells

In the left rat atrium changes in diastolic potential (E max) evoked by sudden stimulation train were modified by adrenaline and ouabain. The early stimulation depolarization phase (SDP) of E max occurring on stimulation was shortened by adrenaline, but lengthened and strongly enhanced by ouabain. The stimulation repolarization phase (SRP) following SDP was markedly inhibited by ouabain, while accelerated and increased by adrenaline. In continuously stimulated atria E max was decreased by ouabain and augmented by adrenaline. The adrenaline-induced hyperpolarization was reduced or suppressed in the presence of 10-4 M or 10-3 M ouabain, respectively. The present data suggest that adrenaline could stimulate the electrogenic sodium pump in the rat atrium.     

Altered electrical response to caffeine exposure in hypertrophied rat myocardium

We investigated the electrophysiological effects of cardiac hypertrophy induced by different experimental models. Comparison of the action potentials of hypertrophied and control rat hearts reveals a pronounced prolongation of the action potential for all types of hypertrophy. This prolongation affects the entire repolarization phase of the action potential 8 days after severe aortic constriction, after 8 days of isoproterenol treatment (5 mg/kg per day), and 3 months after an aortocaval fistula. The electrical changes associated with myocardial hypertrophy induced by pressure overload (aortic constriction) were compared with those resulting from volume overload (aortocaval fistula). Our results show that action potential alterations depend on the nature, duration, and severity of the work load. Thus, pressure overload is more potent to induce these modifications. In the hearts subjected to pressure overload, action potential alterations appear more rapidly and are more marked for the same degree of hypertrophy than those of the volume-hypertrophied myocardium. Furthermore, such data also demonstrate that the early alteration of the action potential during the development of compensatory hypertrophy is a prolongation of the later phase of repolarization (phase 3), without prolongation of the other repolarization phases (1 and 2). This change appears 3 days after aortic constriction, 1 month after coronary artery ligation (in the healthy part of the left ventricle), and 1 month after an aortocaval fistula.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of acetylcholine on the mammalian auricle influenced by verapamil

The action of acetylcholine (ACh) and verapamil (VePa) on the action potential (V(t)), phase plane trajectories of V(t) (dV/dt--V(t) -- plot) and isotonic contractions were investigated using an isolated vegal innervated preparation from rabbit atrium (method I) and investigating action potentials from atrial trabeculae by means a modified sucrose gap technique (method II). If the VePa-concentration increases to 4 mg/1 the duration of the action potential decreases at 20 and 90% repolarization (driving frequencies 2 s-1). In the VePa-solutions phase plane trajectories of the action potential did not change significantly. ACh application favours the disappearance of a region in the repolarization phase plane plot showing anomalous rectification (d(--dV/dt)/dV less than 0) both by control conditions and verapamil. The electrotropic ACh-and vagal effects will be unchanged by verapamil. The inotropic ACh-and vegal action (method I) increases by VePa (2 mg/1). The action of ACh and verapamil will be analysed using a mathematical model for reconstructing the repolarization phase of mammalian atrial myocardium action potentials.     

Subepicardial phase 0 block and discontinuous transmural conduction underlie right precordial ST-segment elevation by a SCN5A loss-of-function mutation

Two mechanisms are generally proposed to explain right precordial ST-segment elevation in Brugada syndrome: 1) right ventricular (RV) subepicardial action potential shortening and/or loss of dome causing transmural dispersion of repolarization; and 2) RV conduction delay. Here we report novel mechanistic insights into ST-segment elevation associated with a Na(+) current (I(Na)) loss-of-function mutation from studies in a Dutch kindred with the COOH-terminal SCN5A variant p.Phe2004Leu. The proband, a man, experienced syncope at age 22 yr and had coved-type ST-segment elevations in ECG leads V1 and V2 and negative T waves in V2. Peak and persistent mutant I(Na) were significantly decreased. I(Na) closed-state inactivation was increased, slow inactivation accelerated, and recovery from inactivation delayed. Computer-simulated I(Na)-dependent excitation was decremental from endo- to epicardium at cycle length 1,000 ms, not at cycle length 300 ms. Propagation was discontinuous across the midmyocardial to epicardial transition region, exhibiting a long local delay due to phase 0 block. Beyond this region, axial excitatory current was provided by phase 2 (dome) of the M-cell action potentials and depended on L-type Ca(2+) current ("phase 2 conduction"). These results explain right precordial ST-segment elevation on the basis of RV transmural gradients of membrane potentials during early repolarization caused by discontinuous conduction. The late slow-upstroke action potentials at the subepicardium produce T-wave inversion in the computed ECG waveform, in line with the clinical ECG.     

Oscillatory zones and their role in normal and abnormal sheep purkinje fiber automaticity

The mechanisms by which low [K(+)](o) induces spontaneous activity was studied in sheep Purkinje fibers. Purkinje strands were superfused in vitro and membrane potentials were recorded by means of a microelectrode technique. The results show that low [K(+)](o) increases the slope and amplitude of early diastolic depolarization, sharpens the transition between early and late diastolic depolarizations, induces an after-potential and large pre-potentials through a negative shift of an oscillatory zone. Pre-potentials occur progressively sooner during diastole and merge with the after-potential to induce uninterrupted spontaneous discharge. During recovery, when the rate slows, after- and pre-potentials separate once more, the slower discharge decreasing the after-potentials but not the pre-potentials. Low [K(+)](o) has little effect on the plateau, but markedly slows phase 3 repolarization and may altogether prevent it. At depolarized levels, voltage oscillations, slow responses, sinusoidal fluctuations or quiescence may be present depending on voltage. During the recovery, a train of either sub-threshold oscillations or spontaneous action potentials appear towards the end of phase 3 repolarization. The cessation of the action potentials unmasks large sub-threshold oscillations, that occur in the oscillatory zone. Drive, high [Ca(2+)](o) and norepinephrine increase slope and amplitude of early diastolic depolarization as low [K(+)](o) does. In low [K(+)](o), Cs(+) prevents spontaneous discharge at polarized levels, but not the decrease in resting potential nor the onset of slow responses at depolarized levels. Cs(+) blocks the train of oscillations and of action potentials occurring during recovery. We conclude that low [K(+)](o) steepens early diastolic depolarization and increases its amplitude through an after-potential that results from an increased Ca(2+) load; allows the attainment of the threshold through Cs(+)-sensitive voltage oscillations which develop when the oscillatory zone is entered either by diastolic depolarization or by phase 3 repolarization; and causes voltage oscillations also at depolarized levels, but through a Cs(+)-insensitive different mechanism.     

Enhanced autophagy plays a cardinal role in mitochondrial dysfunction in type 2 diabetic Goto-Kakizaki (GK) rats: ameliorating effects of (-)-epigallocatechin-3-gallate

Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model--the Goto-Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (-)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.     

Electrical properties of developing rat heart. Effects of dexamethasone

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.     

The effects of bradykinin on electrobiological phenomena in isolated right atrium of the rat.

Bradykinin induces or accelerates the already present electrobiological activity in isolated right atrium of rats. Acceleration of the electrobiological activity induced by bradykinin decreases with increase of initial activity. Bradykinin disturbs transiently the course of the repolarization period in action potential. It may be one of the mechanisms of arrhythmia development caused by bradykinin.     

Nonmitogenic CD3 antibody reverses virally induced (rat insulin promoter-lymphocytic choriomeningitis virus) autoimmune diabetes without impeding viral clearance.

Treatment with nonmitogenic CD3 Ab reverses established autoimmune diabetes in nonobese diabetic mice by restoring self-tolerance, and is currently under clinical evaluation in patients presenting recent onset type I diabetes. Due to the immunosuppressive potential of this strategy, it was relevant to explore how this treatment would influence the outcome of concomitant viral infections. In this study, we used a transgenic model of virally induced autoimmune diabetes (rat insulin promoter-lymphocytic choriomeningitis virus) that allows for more precise tracking of the autoaggressive response and choice of the time point for initiation of autoimmunity. CD3 was most effective during a clearly defined prediabetic phase and prevented up to 100% of diabetes by drastically lowering activation of autoaggressive CD8 lymphocytes and their production of inflammatory cytokines. Interestingly, reversion of established disease could be achieved as well, when nonmitogenic CD3 was administered late during pathogenesis to overtly diabetic recipients. Most importantly, competence to clear viral infections was maintained. Thus, administration of nonmitogenic CD3 prevents diabetes by sufficient systemic reduction of (auto)aggressive lymphocytes, but without compromising antiviral immune competence.     

Gene expression profiles of peripheral blood cells in type 2 diabetes and nephropathy in Asian Indians

Background  

Asian Indians with type 2 diabetes mellitus (T2D) have higher susceptibility to diabetic nephropathy (T2DN), the leading cause of end-stage renal disease and morbidity in diabetes. Peripheral blood cells (PBCs) play an important role in diabetes, yet very little is known about the molecular mechanisms of PBCs regulated in insulin homeostasis. In this study we explored the global gene expression changes in PBCs in diabetes and diabetic nephropathy to identify the potential candidate genes and molecular networks regulated in diabetes and nephropathy.     

Glomerular hemodynamic and structural alterations in experimental diabetes mellitus

Elevated glomerular filtration rate (GFR) is a frequent finding in patients with early insulin-dependent diabetes mellitus (IDDM). The mechanisms responsible for this glomerular hyperfiltration in IDDM are unclear. Rats made diabetic with alloxan or streptozotocin, and treated daily with supplemental insulin, have moderate hyperglycemia and elevated GFR, and thus have been used to study mechanisms of glomerular hyperfiltration in diabetes. Renal micropuncture techniques have shown that single-nephron GFR (SNGFR) is elevated in moderately hyperglycemic diabetic rats. In some cases, this is because of elevated glomerular capillary pressure (Pgc), but in other cases, Pgc is normal despite elevated SNGFR. Several potential mediators of increased SNGFR have been examined, including hyperglycemia, increased glomerular prostaglandin production, and decreased sensitivity of the tubuloglomerular feedback mechanism. Renal failure is a common complication of human IDDM. Diabetic rats with long-term moderate hyperglycemia have been used to study the mechanism by which glomerular injury develops in diabetes mellitus. It has been postulated that glomerular hyperfiltration or some determinant of elevated GFR in early diabetes may ultimately cause glomerular damage, leading to a progressive loss of renal function (diabetic nephropathy). Diabetic rats with long-term moderate hyperglycemia, however, do not develop characteristic glomerular lesions of human diabetic nephropathy and, in fact, develop only minimal glomerular injury even after 1 year of diabetes. Thus, although the diabetic rat with moderate hyperglycemia may be useful to study the mechanisms of glomerular hyperfiltration in early diabetes, it may not be an appropriate model of renal failure in IDDM.     

A Mathematical Model of the Electrophysiological Alterations in Rat Ventricular Myocytes in Type-I Diabetes

Our mathematical model of the rat ventricular myocyte (Pandit et al., 2001) was utilized to explore the ionic mechanism(s) that underlie the altered electrophysiological characteristics associated with the short-term model of streptozotocin-induced, type-I diabetes. The simulations show that the observed reductions in the Ca²⁺-independent transient outward K⁺ current (I_t) and the steady-state outward K⁺ current (I_ss), along with slowed inactivation of the L-type Ca²⁺ current (I_CaL), can result in the prolongation of the action potential duration, a well-known experimental finding. In addition, the model demonstrates that the slowed reactivation kinetics of I_t in diabetic myocytes can account for the more pronounced rate-dependent action potential duration prolongation in diabetes, and that a decrease in the electrogenic Na⁺-K⁺ pump current (I_NaK) results in a small depolarization in the resting membrane potential (V_rest). This depolarization reduces the availability of the Na⁺ channels (I_Na), thereby resulting in a slower upstroke (dV/dt_max) of the diabetic action potential. Additional simulations suggest that a reduction in the magnitude of I_CaL, in combination with impaired sarcoplasmic reticulum uptake can lead to a decreased sarcoplasmic reticulum Ca²⁺ load. These factors contribute to characteristic abnormal [Ca²⁺]_i homeostasis (reduced peak systolic value and rate of decay) in myocytes from diabetic animals. In combination, these simulation results provide novel information and integrative insights concerning plausible ionic mechanisms for the observed changes in cardiac repolarization and excitation-contraction coupling in rat ventricular myocytes in the setting of streptozotocin-induced, type-I diabetes.     

Dynamics of the inward rectifier K+ current during the action potential of guinea pig ventricular myocytes.

The potassium selective, inward rectifier current (IK1) is known to be responsible for maintaining the resting membrane potential of quiescent ventricular myocytes. However, the contribution of this current to the different phases of the cardiac action potential has not been adequately established. In the present study, we have used the action potential clamp (APC) technique to characterize the dynamic changes of a cesium-sensitive (i.e., Ik1) current which occur during the action potential. Our results show that (a) Ik1 is present during depolarization, as well as in the final phase of repolarization of the cardiac action potential. (b) The current reaches the zone of inward-going rectification before the regenerative action potential ensues. (c) The maximal outward current amplitude during repolarization is significantly lower than during depolarization, which supports the hypothesis that in adult guinea pig ventricular myocytes, Ik1 rectification is accentuated during the action potential plateau. Our results stress the importance of Ik1 in the modulation of cell excitability in the ventricular myocyte.     

静脉注射核因子NF-E2相关因子表达质粒减轻糖尿病小鼠肾小球氧化应激损伤的实验研究

该实验旨在研究经小鼠尾静脉快速注射核因子NF-E2相关因子(nuclear factor erythroid2-related factor 2,Nrf2)表达质粒对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病小鼠肾小球氧化应激损伤的保护作用。采用腹腔注射STZ诱发糖尿病小鼠模型,自成模后第3天开始,尾静脉快速注射pcDNA3/mNrf2质粒。4周后收取标本,检测动物肾小球丙二醛(malondialdehyde,MDA)含量,纤维连接蛋白(fibronectin,FN)以及Nrf2、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthethase,γ-GCS)在肾小球的表达。实验结果表明,尾静脉注射可以将Nrf2表达质粒转染入小鼠肾小球。此方法可以降低糖尿病小鼠肾小球MDA浓度,减轻FN在肾小球的表达,增加Nrf2在肾小球细胞核的积聚以及γ-GCS的转录和表达。该研究证明,应用尾静脉注射Nrf2表达质粒的方法可以减轻糖尿病小鼠肾小球氧化应激损伤,减少细胞外基质(extracellular matrix,ECM)沉积,其机制部分是通过激活Nrf2-ARE信号通路而实现的。     

Evaluation of the microsomal glutathione S-transferase 3 (MGST3) locus on 1q23 as a Type 2 diabetes susceptibility gene in Pima Indians

Elevation of plasma glucose concentration may induce generation of oxygen-free radicals, which can play an important role in the progression of diabetes and/or development of its complications. Various glutathione transferases utilize the availability of reduced glutathione for the cellular defense against oxygen-free radicals. One such enzyme is microsomal glutathione S-transferase 3 encoded by MGST3, which maps to chromosome 1q23, a region linked to Type 2 diabetes mellitus (T2DM) in Pima Indians, Caucasian, and Chinese populations. We investigated the MGST3 gene as a potential susceptibility gene for T2DM by screening this locus for single nucleotide polymorphisms (SNPs) in diabetic and non-diabetic Pima Indians. We also measured the skeletal muscle MGST3 mRNA level by Real-Time (RT) PCR and its relationship with insulin action in non-diabetic individuals. We identified 25 diallelic variants, most of which, based on their genotypic concordance, could be divided into three distinct linkage disequilibrium (LD) groups. We genotyped unique representative SNPs in selected diabetic and non-diabetic Pima Indians and found no evidence for association with T2DM. Furthermore, inter-individual variation of skeletal muscle MGST3 mRNA was not correlated with differences in insulin action in non-diabetic subjects. We conclude that alterations of MGST3 are unlikely to contribute to T2DM or differences in insulin sensitivity in the Pima Indians.     

Metabolic memory - the implications for diabetic complications

Many important biochemical mechanisms are activated in the presence of high levels of glucose, which occur in diabetes. Large randomised studies have established that early intensive glycaemic control reduces the risk of diabetic complications. This phenomenon has recently been dubbed 'metabolic memory'. It has been suggested that early glycaemia normalisation can halt the hyperglycaemia-induced pathological processes associated with enhanced oxidative stress and glycation of cellular proteins and lipids. The phenomenon of metabolic memory suggests that early aggressive treatment and strict glycaemic control could prevent chronic diabetic complications.     

Excitation-contraction coupling in myocardium: implications of calcium release and Na+-Ca2+ exchange

In this paper, we present evidence in support of the hypothesis that electrogenic Na+-Ca2+ exchange is responsible for three phenomena in rat cardiac muscle: the slow repolarization phase of the action potential, the time course of the mechanical recovery process, and the development of triggered arrhythmias. It was shown that the duration of the slow phase of repolarization of the action potential varies in proportion to the Na+ concentration gradient and inversely with the Ca2+ concentration gradient over the cell membrane. This suggested that Na+-Ca2+ exchange can generate a current of sufficient magnitude to maintain the membrane depolarized at a level of -60 mV. The mechanical restitution process of rat cardiac trabeculae was shown to exhibit three phase. The first phase, alpha, probably reflects rapid transport of calcium in the sarcoplasmic reticulum from the uptake sites to the release sites. After the initial increase of force during alpha, force rises further during phase beta and then declines during phase gamma. During all phases, force increases with the extracellular calcium concentration. beta is accelerated by preceding extrasystoles, while an increase of the heart rate causes force to increase at approximately the same rate but to a higher level during phase beta. These observations are compatible with a model in which the sarcoplasmic reticulum sequesters calcium from the cytosol, while the membrane of the sarcoplasmic reticulum is assumed to exhibit also a small leak of calcium into the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monophasic action potentials generated by bidomain modeling as a tool for detecting cardiac repolarization times

Unipolar electrograms (EGs) and hybrid (or unorthodox or unipolar) monophasic action potentials (HMAPs) are currently the only proposed extracellular electrical recording techniques for obtaining cardiac recovery maps with high spatial resolution in exposed and isolated hearts. Estimates of the repolarization times from the HMAP downstroke phase have been the subject of recent controversies. The goal of this paper is to computationally address the controversies concerning the HMAP information content, in particular the reliability of estimating the repolarization time from the HMAP downstroke phase. Three-dimensional numerical simulations were performed by using the anisotropic bidomain model with a region of short action potential durations. EGs, transmembrane action potentials (TAPs), and HMAPs elicited by an epicardial stimulation close or away from a permanently depolarized site were computed. The repolarization time was computed as the moment of EG fastest upstroke (RT(eg)) during the T wave, of HMAP fastest downstroke (RT(HMAP)), and of TAP fastest downstroke (RT(tap)). The latter was taken as the gold standard for repolarization time. We also compared the times (RT90(HMAP), RT90(tap)) when the HMAP and TAP first reach 90% of their resting value during the downstroke. For all explored sites, the HMAP downstroke closely followed the TAP downstroke, which is the expression of local repolarization activity. Results show that HMAP and TAP markers are highly correlated, and both markers RT(HMAP) and RT(eg) (RT90(HMAP)) are reliable estimates of the TAP reference marker RT(tap) (RT90(tap)). Therefore, the downstroke phase of the HMAP contains valuable information for assessing repolarization times.     

The Repolarization Phase of the Cardiac Ventricular Action Potential: A Time-Dependent System of Membrane Conductances

A system for the generation of the repolarization phase of the ventricular action potential is described. The system is based on time-dependent changes in membrane conductance to sodium and potassium ions. However, the changes in conductance during an action potential retain a degree of voltage dependence through the initial conditions which depend on previous depolarizations of the membrane. The equations describing the system were solved with an analog computer and various action potential forms are reproduced. The effects of hyperpolarizing and depolarizing current applied during an action potential are investigated. The changes in shape of an action potential after a change in the rate of stimulation show partial agreement with previous experimental findings. The applicability of time-dependent and voltage-dependent systems for the generation of the repolarization phase of the ventricular action potential is discussed.