Effects of acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin-induced diabetic rats

We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca2+ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35-36 degrees C. At 8-12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca2+ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca2+ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca2+ and altered mechanisms of sarcoplasmic reticulum Ca2+ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat.     

Effects of single high-dose and multiple low-dose streptozotocin on contraction and intracellular Ca<Superscript>2+</Superscript> in ventricular myocytes from diabetes resistant and susceptible rats

Administration of a single high-dose (SHD) of streptozotocin (STZ) to young adult rats causes a diabetic cardiomyopathy. Albino Oxford (AO) and Dark Agouti (DA) inbred strains of rats are susceptible to developing diabetes when administered a SHD of STZ but differ in susceptibility to multiple low-dose (MLD) STZ. We have investigated the effects of SHD and MLD-STZ on contraction and intracellular Ca²⁺, measured with fura-2, in ventricular myocytes from AO and DA rats at 18–20 weeks after treatment. Time to peak shortening was significantly prolonged in myocytes from DA rats after SHD-STZ but was not altered in DA rats after MLD-STZ or in AO rats by either MLD or SHZ-STZ treatment. Time to peak shortening in myocytes from DA control and DA rats after SHD-STZ were 88 ± 2 ms and 107 ± 4 ms, respectively. Time to half relaxation and the amplitude of myocyte shortening were not altered in AO or DA rats by either MLD or SHD-STZ treatment. Amplitude, time to peak fura-2 transient and time to half relaxation of the fura-2 transient were not significantly altered in AO or DA rats by either MLD or SHD-STZ treatment. Contractile defects reported in myocytes from SHD-STZ treated DA rats may be a consequence of altered myofilament sensitivity to Ca²⁺. The hyperglycaemic effects of MLD-STZ and SHD-STZ induced diabetes was much greater in DA compared to AO rats and the effects of the hyperglycaemia on the time-course of ventricular myocyte contraction was most profound in DA rats after SHD-STZ. (Mol Cell Biochem 269: 103–108, 2005)     

Halothane alters contractility and Ca2+ transport in ventricular myocytes from streptozotocin-induced diabetic rats

General anaesthetics have previously been shown to have profound effects on myocardial function. Moreover, many patients suffering from diabetes mellitus are anaesthetised during surgery. This study investigated compromised functioning of cardiac myocytes from streptozotocin (STZ)-induced diabetic rats and the additive effects of halothane on these dysfunctions. Ventricular myocytes were isolated from 8 to 12 weeks STZ-treated rats. Contraction and intracellular free calcium concentration ([Ca²⁺] _i ) were measured in electrically field-stimulated (1 Hz) fura-2-AM-loaded cells using a video-edge detection system and a fluorescence photometry system, respectively. L-type Ca²⁺ current was measured in whole cell, voltage-clamp mode. Halothane significantly (p < 0.01) depressed the amplitude and the time course of the Ca²⁺ transients in a similar manner in myocytes from control and STZ-treated rats. However, the effect of halothane on the amplitude of shortening and L-type Ca²⁺ current was more pronounced in myocytes from STZ-treated animals compared to age-matched controls. Myofilament sensitivity to Ca²⁺ was significantly (p < 0.01) increased in myocytes from STZ-treated rats compared to control. However, in the presence of halothane the myofilament sensitivity to Ca²⁺ was significantly (p < 0.05) reduced to a greater extent in myocytes from STZ-treated rats compared to controls. In conclusion, these results show that contractility, Ca²⁺ transport and myofilament sensitivity were all altered in myocytes from STZ-treated rats and these processes were further altered in the presence of halothane suggesting that hearts from STZ-induced diabetic rats are sensitive to halothane. (Mol Cell Biochem 261: 251–261, 2004)     

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 (I _Ca,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 I _Ca,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)     

Stable microtubules contribute to cardiac dysfunction in the streptozotocin-induced model of type 1 diabetes in the rat

Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular myocytes were isolated from rats at 8–12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes showed a tendency for reduced shortening and intracellular Ca²⁺ ([Ca²⁺] _i ) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca²⁺] _i . Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 μM) than control myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC increased shortening and [Ca²⁺] _i transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased ttp shortening and [Ca²⁺] _i only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes, regulating both [Ca²⁺] _i and shortening. Holly Shiels and Anthony O’Connell are equal first authorship.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

Vitamin E Ameliorates the Decremental Effect of Paraquat on Cardiomyocyte Contractility in Rats

Background

Exposure to pesticides and industrial toxins are implicated in cardiovascular disease. Paraquat (PAR) is a toxic chemical widely used as an herbicide in developing countries and described as a major suicide agent. The hypothesis tested here is that PAR induced myocardial dysfunction may be attributed to altered mechanisms of Ca²⁺ transport which are in turn possibly linked to oxidative stress. The mechanisms of PAR induced myocardial dysfunction and the impact of antioxidant protection was investigated in rat ventricular myocytes.

Methodology

Forty adult male Wistar rats were divided into 4 groups receiving the following daily intraperitoneal injections for 3 weeks: Group 1 PAR (10 mg/kg), Control Group 2 saline, Group 3 vitamin E (100 mg/kg) and Group 4 PAR (10 mg/kg) and vitamin E (100 mg/kg). Ventricular action potentials were measured in isolated perfused heart, shortening and intracellular Ca²⁺ in electrically stimulated ventricular myocytes by video edge detection and fluorescence photometry techniques, and superoxide dismutase (SOD) and catalase (CAT) levels in heart tissue.

Principal Findings

Spontaneous heart rate, resting cell length, time to peak (TPK) and time to half (THALF) relaxation of myocyte shortening were unaltered. Amplitude of shortening was significantly reduced in PAR treated rats (4.99±0.26%) and was normalized by vitamin E (7.46±0.44%) compared to controls (7.87±0.52%). PAR significantly increased myocytes resting intracellular Ca²⁺ whilst TPK and THALF decay and amplitude of the Ca²⁺ transient were unaltered. The fura-2–cell length trajectory during the relaxation of the twitch contraction was significantly altered in myocytes from PAR treated rats compared to controls suggesting altered myofilament sensitivity to Ca²⁺ as it was normalized by vitamin E treatment. A significant increase in SOD and CAT activities was observed in both PAR and vitamin E plus PAR groups.

Conclusions

PAR exposure compromised rats heart function and ameliorated by vitamin E treatment.     

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

Background

There is limited knowledge about atrial myocyte Ca²⁺ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca²⁺ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.

Methods and results

Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca²⁺ amplitude, decreased SR Ca²⁺ content, and slower Ca²⁺ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca²⁺ ATPase activity, increased Na⁺/Ca²⁺-exchanger activity and increased diastolic Ca²⁺ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca²⁺ handling in HF.

Conclusion

Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca²⁺ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.     

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation–contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation–contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10–11 months following 2–3 months of treadmill exercise training. Shortening, [Ca²⁺]_i and L-type Ca²⁺ current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca²⁺ transients and L-type Ca²⁺ current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca²⁺ transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.     

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca²⁺ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca²⁺ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca²⁺ transients. However, the amplitudes of caffeine-evoked Ca²⁺ transients and the rate of recovery of electrically evoked Ca²⁺ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca²⁺-uptake and Ca²⁺-ATPase activity in a biphasic manner. [³H]-ryanodine binding and passive Ca²⁺ release from SR vesicles were not altered by 10 μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 μg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.     

Distribution of atrial natriuretic peptide and its effects on contraction and intracellular calcium in ventricular myocytes from streptozotocin-induced diabetic rat

The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte contraction and intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood plasma concentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to age-matched controls. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myocytes the percentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-treated rat the percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myocytes from STZ-treated rats (360+/-5 ms) compared to controls (305+/-5 ms). Amplitude of the Ca2+ transient was significantly increased in myocytes from STZ-treated rats compared to controls (0.39+/-0.02 versus 0.29+/-0.02 fura-2 RU in controls) and treatment with ANP reduced the amplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.     

Long-term administration of rosuvastatin prevents contractile and electrical remodelling of diabetic rat heart

In recent years, many findings have been presented about the potential benefit of statin therapy on diabetes-induced cardiovascular complications. Cardioprotective effects of statins were suggested to be mediated at least in part through inhibition of small GTPases, particularly those of the Rho family. The present study was designed to examine whether rosuvastatin can improve electrical remodeling and contractile dysfunction in type 1 diabetic rat heart via modulation of RhoA pathway. Type 1 diabetes was induced by single dose injection of STZ (50 mg/kg). One week after injection rosuvastatin (10 mg/kg/day) and sham treatment was given for 5 weeks in the diabetic rats, as well as in control groups. Shortening and Ca²⁺ transients were recorded in myocytes loaded with Fura2-AM. Membrane currents and Ca²⁺ transients were measured synchronously via whole-cell patch clamping. In untreated diabetic rats, relaxation of shortening and decay of the matched Ca²⁺ transients were prolonged. Fractional shortening and Ca²⁺ transients were also decreased. Rosuvastatin treatment reversed those changes. I_CaL density did not change in either group but rosuvastatin recovered the loss of sarcoplasmic reticulum Ca²⁺ and Na⁺/Ca²⁺ exchange as evidenced from amplitude and decay of caffeine-induced Ca²⁺ transients, peak I_NCX and calculated sarcoplasmic reticulum Ca²⁺ content. Diabetes-induced attenuation of I_to and I_sus was also reversed, whilst I_K1 was unchanged in diabetes and unaffected by treatment. Rosuvastatin prevented the diabetes-induced increase in RhoA expression. Plasma cholesterol and triglyceride levels were higher in diabetic rats, but rosuvastatin reduced only the latter. In conclusion, HMG-CoA reductase inhibitor rosuvastatin can prevent diabetes-induced electrical and functional remodeling of heart due to inhibition of RhoA signalling rather than reduction of cholesterol level.     

Effects of single high-dose and multiple low-dose streptozotocin on contraction and intracellular Ca2+ in ventricular myocytes from diabetes resistant and susceptible rats

Administration of a single high-dose (SHD) of streptozotocin (STZ) to young adult rats causes a diabetic cardiomyopathy. Albino Oxford (AO) and Dark Agouti (DA) inbred strains of rats are susceptible to developing diabetes when administered a SHD of STZ but differ in susceptibility to multiple low-dose (MLD) STZ. We have investigated the effects of SHD and MLD-STZ on contraction and intracellular Ca2+, measured with fura-2, in ventricular myocytes from AO and DA rats at 18-20 weeks after treatment. Time to peak shortening was significantly prolonged in myocytes from DA rats after SHD-STZ but was not altered in DA rats after MLD-STZ or in AO rats by either MLD or SHZ-STZ treatment. Time to peak shortening in myocytes from DA control and DA rats after SHD-STZ were 88+/-2 ms and 107+/-4 ms, respectively. Time to half relaxation and the amplitude of myocyte shortening were not altered in AO or DA rats by either MLD or SHD-STZ treatment. Amplitude, time to peak fura-2 transient and time to half relaxation of the fura-2 transient were not significantly altered in AO or DA rats by either MLD or SHD-STZ treatment. Contractile defects reported in myocytes from SHD-STZ treated DA rats may be a consequence of altered myofilament sensitivity to Ca2+. The hyperglycaemic effects of MLD-STZ and SHD-STZ induced diabetes was much greater in DA compared to AO rats and the effects of the hyperglycaemia on the time-course of ventricular myocyte contraction was most profound in DA rats after SHD-STZ.     

The progressive effects of a fat enriched diet on ventricular myocyte contraction and intracellular Ca2+ in the C57BL/6J mouse

The C57BL/6J mouse has a genetic susceptibility to develop diabetes when fed with a high-fat, high-sucrose diet. The general characteristics of diet-induced diabetes in this model include progressive development of hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity, features that are frequently observed in the clinical setting. This study investigated the progressive effects of a fat enriched (FE) diet on contraction and intracellular Ca²⁺ in ventricular myocytes from the C57BL/6J mouse. The characteristics of the mice fed with the FE diet compared to mice receiving control diet included progressive increase in the rate of body weight gain, increased fasting blood glucose and time-dependent differences in the disposal of blood glucose after a glucose challenge. The ultrastructure of cardiac myocytes and associated capillaries did not show any gross morphological alteration after 27 weeks of FE diet compared to controls.At 5 months the resting cell length (RCL) and the kinetics of shortening were not significantly altered in ventricular myocytes from mice receiving the FE diet compared to age-matched controls. At 5 and at 7 months the amplitude of shortening was increased in myocytes receiving the FED diet compared to controls. At 7 months the time to half (THALF) relaxation of myocyte contraction was shortened in myocytes from mice receiving the FE diet compared to controls. Mean THALF relaxation in myocytes from mice fed the FE diet was 32.0 ± 1.4 ms (n = 23) compared to 40.2 ± 2.0 ms (n = 27) in controls. Neither resting intracellular Ca²⁺ nor the kinetics or amplitude of the Ca²⁺ transient were altered by FE diet. Differences in myofilament sensitivity to Ca²⁺ might underlie the changes in contractility.     

Interpretation of relevance of sodium–calcium exchange in action potential of diabetic rat heart by mathematical model

Sarcolemmal Na⁺–Ca²⁺ exchange plays a central role in ion transport of the myocardium and the current carried with it contributes to the late phase of the action potential (AP) besides the contribution of outward K⁺-currents. In this study, the mathematical model for AP of the diabetic rat ventricular myocytes [34] was modified and used for the diabetic rat papillary muscle. We used our experimentally measured values of two K⁺-currents; transient outward current, I_to and steady-state outward current, I_ss, as well as L-type Ca²⁺-current, I_CaL, then compared with the simulated values. We have demonstrated that the prolongation in the AP of the papillary muscle of the diabetic rats are not due to the alteration of I_CaL but mainly due to the inhibition of the K⁺-currents and also the Na⁺–Ca²⁺ exchanger current, I_Na–Ca. In combination with our experimental data on sodium-selenite-treated diabetic rats, our simulation results provide new information concerning plausible ionic mechanisms, and second a possible positive effect of selenium treatment on the altered I_Na–Ca for the observed changes in the AP duration of streptozotocin-induced diabetic rat heart. (Mol Cell Biochem 269: 121–129, 2005)     

Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced diabetes mellitus in rat: Effect of cholecystokinin-octapeptide

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca²⁺) and magnesium (Mg²⁺) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg^–1, I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg^–1 urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca²⁺ and Mg²⁺ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 ± 2.42 mg dl^–1 (n= 44) and >500 mg dl^–1 (n= 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 ± 0.05 ul min^–1 (n= 10) and 1.28 ± 0.16 ul min^–1 (n= 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca²⁺]_i in control and diabetic fura-2-loaded acinar cells was 109.40 ± 15.41 nM (n= 15) and 130.62 ± 17.66 nM (n= 8), respectively. CCK-8 (10^–8M) induced a peak response of 436.55 ± 36.54 nM (n= 15) and 409.31 ± 34.64 nM (n= 8) in control and diabetic cells, respectively. Basal [Mg²⁺]_i in control and diabetic magfura-2-loaded acinar cells was 0.96 ± 0.06 nM (n= 18) and 0.86 ± 0.04 nM (n= 10). In the presence of CCK-8 (10^–8) [Mg²⁺]_i in control and diabetic cells was 0.80 ± 0.05 nM (n= 18) and 0.60 ± 0.02 nM (n= 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca²⁺ and Mg²⁺ homeostasis. (Mol Cell Biochem 261: 83–89, 2004)     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

Characterisation of ventricular myocyte shortening after administration of streptozotocin (STZ) to neonatal rats

Human and animal studies have shown that diabetes mellitus can be associated with altered cardiac function that is independent of vascular complications. Streptozotocin (STZ) is a diabetogenic agent and when administered to rats causes selective beta-cell necrosis which is accompanied by a drastic reduction in plasma insulin and hyperglycaemia. We have investigated the characteristics of shortening in ventricular myocytes isolated from rat heart at 10 months after administration of STZ to neonatal rats at 2 days of age. The characteristics of shortening in myocytes from the STZ-treated neonatal rat compared to shortening in myocytes from the STZ-treated young adult rat are discussed. STZ-treated rats gained significantly less weight compared to age-matched controls. Although non-fasting blood glucose was not significantly different in STZ-treated rats they were found to be markedly glucose intolerant when given an intraperitoneal challenge of glucose (2 g/kg) after an overnight fast. During electrical stimulation (1 or 2 Hz) ventricular myocyte resting length, time to peak shortening, time to half relaxation and amplitude of shortening were not altered after STZ treatment. The imposition of rest periods (2-60 s) after trains of electrically stimulated (1 Hz) steady-state contractions resulted in a potentiation of the contraction, which immediately followed the rest period (post rest potentiation). Post rest potentiation was larger, following rest periods between 20 and 60 s, in myocytes from STZ-treated rats compared to controls. The absence of major alterations to the amplitude and kinetics of contraction in myocytes from STZ-treated neonatal rats at 10 months after treatment might be explained by a partial recovery of the beta-cells in the growing animal.     

Effects of halothane,isoflurane, sevoflurane and desflurane on contraction of ventricular myocytes from streptozotocin-induced diabetic rats

Various clinically used volatile general anaesthetics (e.g. sevoflurane, halothane, isoflurane and desflurane) have been shown to have significant negative inotropic effects on normal ventricular muscle. However, little is known about their effects in ventricular tissue from diabetic animals. Streptozotocin (STZ)-induced diabetes is known to induce changes in the amplitude and time course of shortening and one report suggests that the inotropic effects of anaesthetics are ameliorated in papillary muscles from diabetic animals. The aim of these studies was to investigate this further in electrically stimulated (1 Hz) ventricular myocytes. Cells were superfused with either normal Tyrode (NT) solution or NT containing anaesthetic (1 mM) for a period of 2 min (at 30–32°C). Myocytes from STZ rats were shown to have a significantly longer time to peak shortening (p > 0.001, n= 50) and the amplitude of shortening tended to be greater but this was not significant (p= 0.13, n= 50). Halothane, isoflurane, desflurane and sevoflurane significantly (p < 0.05) reduced the magnitude of shortening of control cells by 72.5 ± 3.2%, 46.5 ± 9.7%, 28.9 ± 4.3% and 22.8 ± 5.6%, respectively (n > 11 per group) but their steady-state negative inotropic effect was found to be no different in cells from STZ-treated rats (73.0 ± 4.8%, 40.7 ± 4.7%, 25.0 ± 5.2% and 19.8 ± 5.2%, respectively, n > 10 per group). Therefore, we conclude that the inotropic effects of volatile anaesthetics were not altered by STZ treatment. (Mol Cell Biochem 261: 209–215, 2004)     

The effect of insulin-like growth factor-1 on adult rat cardiac contractility

There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-I on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca²⁺) handling in isolated cardiac myocytes. IGF-I had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 ± 4.6%, 18.5 ± 5.8% and 11.9 ± 4.9% (n = 12–20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 ± 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 ± 3.2%, 17.7 ± 4.1% and 19.7 ± 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16–20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca²⁺ concentration, the amplitude of the Ca²⁺ transient and the time to peak Ca²⁺ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca²⁺ in isolated cardiac myocytes. Increasing insulin concentrations (1–1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca²⁺. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca²⁺ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.