Frank Christopher Howarth

Long‐term effects of streptozotocin‐induced diabetes on the electrocardiogram, physical activity and body temperature in rats

In vivo biotelemetry studies have demonstrated that short‐term streptozotocin (STZ)‐induced diabetes is associated with a reduction in heart rate (HR) and heart rate variability (HRV) and prolongation of QT and QRS intervals. This study investigates the long‐term effects of STZ‐induced diabetes on the electrocardiogram (ECG), physical activity and body temperature. Transmitter devices were surgically implanted in the peritoneal cavity of young adult male Wistar rats. Electrodes from the transmitter were arranged in Einthoven bipolar lead II configuration. ECG, physical activity and body temperature data were continuously recorded with a telemetry system before and following the administration of STZ (60 mg kg−1) for a period of 22 weeks. HR, physical activity and body temperature declined rapidly 3–5 days after the administration of STZ. The effects became conspicuous with time reaching a new steady state approximately 1–2 weeks after STZ treatment. HR at 4 weeks was 268 ± 5 beats min−1 in diabetic rats compared to 347 ± 12 beats min−1 in age‐matched controls. HRV at 4 weeks was also significantly reduced after STZ treatment (18 ± 3 beats min−1) compared to controls (33 ± 3 beats min−1). HR and HRV were not additionally altered in either diabetic rats (266 ± 5 and 20 ± 4 beats min−1) or age‐matched controls (316 ± 6 and 25 ± 4 beats min−1) at 22 weeks. Reduced physical activity and/or body temperature may partly underlie the reductions in HR and HRV. In addition, the increased power spectral low frequency/high frequency ratio from 4 weeks after STZ treatment may indicate an accompanying disturbance in sympathovagal balance.

A method for making rapid changes of superfusate whilst maintaining temperature at 37°C

We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37°C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37°C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min−1, linear flow velocity 11.6 cm s−1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3°C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature.

Effect of the microtubule polymerizing agent taxol on contraction, Ca2+ transient and L-type Ca2+ current in rat ventricular myocytes

1 Microtubules form part of the cytoskeleton. Their role in adult ventricular myocytes is not well understood although microtubule proliferation has previously been linked with reduced contractile function. 2 We investigated the effect of the anti‐tumour drug taxol, a known microtubule polymerizing agent, on Ca2+ handling in adult rat ventricular myocytes. 3 Treatment of cells with taxol caused proliferation of microtubules. 4 In taxol‐treated cells there was a reduction in the amplitude of contraction, no significant effect on the amplitude of L‐type Ca2+ current, but a significant reduction in the amplitude of the Ca2+ transient. 5 Caffeine was used to release Ca2+ from the sarcoplasmic reticulum (SR). There was a significant reduction in the ratio of electrically stimulated : caffeine‐induced Ca2+ transients in taxol‐treated cells. This observation is consistent with the hypothesis that taxol reduces fractional SR Ca2+ release. 6 We suggest that the negative inotropic effect of taxol may, at least in part, be the result of reduced release of Ca2+ from the SR. Microtubules may be important regulators of Ca2+ handling in the heart.

Pathogenesis and pathophysiology of accelerated atherosclerosis in the diabetic heart

It has been established that atherosclerotic coronary artery disease is more frequent and more severe in diabetic compared to non-diabetic subjects, but the reason for the excess risk of developing coronary macroangiopathy in diabetes remains incompletely characterized. Various biochemical mechanisms speculated to being at the “heart” of diabetic cardiac and coronary macroangiopathy are reviewed in the present article. In doing so, this article presents evidence that the labyrinthine interactions of hyperglycemia, insulin resistance, and dyslipidemia in diabetes result in a pro-atherogenic phenotype. Furthermore, the diabetic milieu yields a complex (dys)metabolic environment characterized by chronic inflammation, procoagulability, impaired fibrinolysis, neovascularization abnormalities, and microvascular defects that cumulatively alter blood rheology, artery structure, and homeostasis of the endothelium. The contributory influences of these factors in the pathophysiology of coronary artery disease in diabetes are also discussed.

Characteristics of the delayed rectifier K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart.

The delayed rectifier potassium current (IK) is known to be important in action potential repolarisation and may contribute to the diastolic pacemaker depolarisation in pacemaker cells from the heart. In this study, using whole-cell patch clamp, we investigated the characteristics ofIK in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at −40 mV and 5 μM external nifedipine was used to block L-type calcium current (ICa,L). SignificantIK was observed with pulses to potentials more positive than −30 mV. The steady-state activation curve in both cell types showed maximal activation at between + 10 and + 20 mV. Half-maximal activation ofIK occurred at −4.9 and −4.1 mV with slope factors of 8.3 and 12.4 mV in ventricular and AVN cells, respectively. Using pulses of increasing duration, significantIK tails after repolarisation from + 40 mV were observed with pulses of 20 ms and increased with pulses up to 100–120 ms in both cell types. Pulses of longer duration did not activate furtherIK and this suggested that only the rapid component ofIK, calledIKr, was present in either cell type. Moreover,IK tails after pulses to all potentials were blocked completely by E-4031, a selective blocker ofIKr. The reversal potential ofIK varied with the concentration of external K. Superfusion of AVN cells with medium containing 4, 15 and 40 mM [K+]o resulted in reversal potentials of −81, −56 and −32 mV respectively, which are close to values predicted if theIK channel were highly selective for K. The time constants for deactivation ofIK in ventricle and AVN on return to −40 mV after a 500-ms activating pulse to + 60 mV were 480 ms and 230 ms, respectively. The faster deactivation ofIK in AVN cells was a distinguishing feature and suggests that there may be differences in theIKr channel protein between ventricular and AVN cells.

Increase in neuronal nitric oxide synthase content of the gastroduodenal tract of diabetic rats.

Abstract: This study examined the changes occurring in the pattern of distribution and expression of neuronal nitric oxide synthase (nNOS)-positive nerves in the gastroduodenal tract of streptozotocin-induced diabetic rats. The ganglion cells of the myenteric plexus of the gastric antrum of normal rats contain nNOS. We also observed nNOS-positive neurons and fibres in the myenteric plexus of the duodenum of normal rats. After the onset of diabetes, the number and intensity of staining of nNOS-positive nerve profiles in the gastric antrum and duodenum did not change significantly. However, Western blotting showed a significant increase in the expression of nNOS after the onset of diabetes. In conclusion, diabetes of 4 and 32 weeks duration induced an increase in the tissue content of nNOS in the gastroduodenum of rat. The increase in the level of nNOS in the gastroduodenum of diabetic rats may explain why impaired gastric emptying is common in patients with diabetes.

Increased Oxidative Stress and Mitochondrial Dysfunction in Zucker Diabetic Rat Liver and Brain

Background/Aims: The Zucker diabetic fatty (ZDF, FA/FA) rat is a genetic model of type 2 diabetes, characterized by insulin resistance with progressive metabolic syndrome. We have previously demonstrated mitochondrial dysfunction and oxidative stress in the heart, kidneys and pancreas of ZDF rats. However, the precise molecular mechanism of disease progression is not clear. Our aim in the present study was to investigate oxidative stress and mitochondrial dysfunction in the liver and brain of ZDF rats. Methods: In this study, we have measured mitochondrial oxidative stress, bioenergetics and redox homeostasis in the liver and brain of ZDF rats. Results: Our results showed increased reactive oxygen species (ROS) production in the ZDF rat brain compared to the liver, while nitric oxide (NO) production was markedly increased both in the brain and liver. High levels of lipid and protein peroxidation were also observed in these tissues. Glutathione metabolism and mitochondrial respiratory functions were adversely affected in ZDF rats when compared to Zucker lean (ZL, +/FA) control rats. Reduced ATP synthesis was also observed in the liver and brain of ZDF rats. Western blot analysis confirmed altered expression of cytochrome P450 2E1, iNOS, p-JNK, and IκB-a confirming an increase in oxidative and metabolic stress in ZDF rat tissues. Conclusions: Our data shows that, like other tissues, ZDF rat liver and brain develop complications associated with redox homeostasis and mitochondrial dysfunction. These results, thus, might have implications in understanding the etiology and pathophysiology of diabesity which in turn, would help in managing the disease associated complications.

Left ventricle structural remodelling in the prediabetic Goto–Kakizaki rat

This study tested the hypothesis that experimental prediabetes can elicit structural remodelling in the left ventricle (LV). Left ventricles isolated from 8‐week‐old male Goto–Kakizaki (GK) rats and age‐matched male Wistar control rats were used to assess remodelling changes and underlying transforming growth factor β1 (TGFβ1) activity, prohypertrophic Akt–p70S6K1 signalling and gene expression profile of the extracellular matrix (ECM) using histological, immunohistochemical, immunoblotting and quantitative gene expression analyses. Prediabetes in GK rats was confirmed by impaired glucose tolerance and modestly elevated fasting blood glucose. Left ventricle remodelling in the GK rat presented with marked hypertrophy of cardiomyocytes and increased ECM deposition that together translated into increased heart size in the absence of ultrastructural changes or fibre disarray. Molecular derangements underlying this phenotype included recapitulation of the fetal gene phenotype markers B‐type natriuretic peptide and α‐skeletal muscle actin, activation of the Akt–p70S6K1 pathway and altered gene expression profile of key components (collagen 1α and fibronectin) and modulators of the ECM (matrix metalloproteinases 2 and 9 and connective tissue growth factor). These changes were correlated with parallel findings of increased TGFβ1 transcription and activation in the LV and elevated active TGFβ1 in plasma of GK rats compared with control animals (Students t test, P < 0.05 versus age‐matched Wistar control animals for all parameters). This is the first report to describe LV structural remodelling in experimental prediabetes. The results suggest that ventricular decompensation pathognomonic of advanced diabetic cardiomyopathy may have possible origins in profibrotic and prohypertrophic mechanisms triggered before the onset of type 2 diabetes mellitus.

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+ signalling in young type 2 Zucker diabetic fatty rat heart

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca2+ transport in young (9‐ to 13‐week‐old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time‐to‐peak shortening and time to half‐relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age‐matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca2+ transient was unaltered; however, time‐to‐peak Ca2+ transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L‐type Ca2+ current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L‐type Ca2+ current was 1.19 ± 0.28 pA pF−1 in ZDF myocytes compared with 2.42 ± 0.40 pA pF−1 in control myocytes. Sarcoplasmic reticulum Ca2+ content, release and uptake and myofilament sensitivity to Ca2+ were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L‐type Ca2+ channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca2+ transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L‐type Ca2+ channel proteins might partly underlie alterations in the time course of contraction and Ca2+ transients in ventricular myocytes from ZDF rats.

Voltage-dependence of contraction in streptozotocin-induced diabetic myocytes.

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of −40 mV, test pulses were applied at potentials between −30 and +50 mV in 10 mV increments. L-type Ca2+ current (ICa,L) density and contraction were measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The ICa,L density was significantly (p < 0.05) reduced throughout voltage ranges (−10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between −20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca2+ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca2+ trigger through L-type-Ca2+-channels and lead to normalised contraction. (Mol Cell Biochem 261: 235–243, 2004)