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.     

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)     

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°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. (Mol Cell Biochem 261: 227–233, 2004)     

Effects of MCC-135 on Ca2+ uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca2+ in isolated ventricular muscles of rats with diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca²⁺. Although MCC-135, an intracellular Ca²⁺-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca²⁺ uptake by SR and myofilament sensitivity to Ca²⁺. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca²⁺ uptake and myofilament sensitivity to Ca²⁺, respectively. In diabetic rats, SR Ca²⁺ uptake was decreased, which was related to decrease in protein level of SR Ca²⁺-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca²⁺ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca²⁺ was increased, without significant change in myofilament sensitivity to Ca²⁺ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca²⁺ in diabetic rats. These results suggest that MCC-135 enhances SR Ca²⁺ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca²⁺. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca²⁺ sparks are the elementary events of intracellular Ca²⁺ release from the sarcoplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca²⁺ channel activation contributes to the genesis of spontaneous Ca²⁺ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca²⁺ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI₂ which inhibits spontaneous L-type Ca²⁺ channel activation, the rate of occurrence of spontaneous Ca²⁺ sparks was halved from 4.20 to 2.04 events/(100 μm · s), with temporal and spatial properties of individual Ca²⁺ sparks unchanged. Analysis of the Cd²⁺-sensitive spark production revealed an open probability of ~10 ^-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca²⁺ channels in resting heart cells contribute significantly to the production of spontaneous Ca²⁺ sparks.     

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.     

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)     

Modified Cytoplasmic Ca2+ Sequestration Contributes to Spinal Cord Injury-Induced Augmentation of Nerve-Evoked Contractions in the Rat Tail Artery

In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca²⁺ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca²⁺ entering via L-type Ca²⁺ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca²⁺ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca²⁺ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca²⁺ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca²⁺ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca²⁺ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.     

The alkaloid matrine of the root of Sophora flavescens prevents arrhythmogenic effect of ouabain

Matrine, a alkaloid of the root of Sophora flavescens, has multiple protective effects on the cardiovascular system including cardiac arrhythmias. However, the molecular and ionic mechanisms of matrine have not been well investigated. Our study aimed at to shed a light on the issue to investigate the antiarrhythmic effects of matrine by using ouabain to construct an arrhythmic model of cardiomyocytes. In this experiment, matrine significantly and dose-dependently increased the doses of ouabain required to induce cardiac arrhythmias and decreased the duration of arrhythmias in guinea pigs. In cardiomyocytes of guinea pigs, ouabain 10 μM prolonged action potential duration by 80% (p < 0.05) and increased L-type Ca²⁺ currents and Ca²⁺ transients induced by KCl (p < 0.05). Matrine 100 μM shortened the prolongation of APD and prevented the increase of L-type Ca²⁺ currents and Ca²⁺ transients induced by ouabain. Taken together, these findings provide the first evidence that matrine possessed arrhythmogenic effect of ouabain by inhibiting of L-type Ca²⁺ currents and Ca²⁺ overload in guinea pigs.     

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.     

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.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A

Viral-mediated gene transfer of troponin I(TnI) isoforms and chimeras into adult rat cardiac myocytes was used toinvestigate the role TnI domains play in the myofilament tensionresponse to protein kinase A (PKA). In myocytes expressing endogenouscardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein Cand decreased the Ca²⁺ sensitivity of myofilament tension.In marked contrast, PKA did not influence Ca²⁺-activatedtension in myocytes expressing the slow skeletal isoform of TnI or achimera (N-slow/card-C TnI), which lack the unique phosphorylatableamino terminal extension found in cTnI. PKA-mediated phosphorylation ofa second TnI chimera, N-card/slow-C TnI, which has the amino terminalregion of cTnI, caused a decrease in the Ca²⁺ sensitivityof tension comparable in magnitude to control myocytes. Based on theseresults, we propose the amino terminal region shared by cTnI andN-card/slow-C TnI plays a central role in determining the magnitude ofthe PKA-mediated shift in myofilament Ca²⁺ sensitivity,independent of the isoform-specific functional domains previouslydefined within the carboxyl terminal backbone of TnI. Interestingly,exposure of permeabilized myocytes to acidic pH after PKA-mediatedphosphorylation of cTnI resulted in an additive decrease in myofilamentCa²⁺ sensitivity. The isoform-specific, pH-sensitive regionwithin TnI lies in the carboxyl terminus of TnI, and the additiveresponse provides further evidence for the presence of a separatedomain that directly transduces the PKA phosphorylation signal.

     

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.     

Rho-kinase inhibition reverses impaired Ca2+ handling and associated left ventricular dysfunction in pressure overload-induced cardiac hypertrophy

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca²⁺ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca²⁺ homeostasis.Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC + Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC + Fas group were administered fasudil (5 mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC + Fas myocytes showed significant improvement in reduced contractility and Ca²⁺ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca²⁺ reuptake. Although L-type Ca²⁺ currents did not change in TAC group, there was a significant reduction in the triggered Ca²⁺ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB^S16/T17/SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression.Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca²⁺ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure.     

Characterizing the influence of chronic hypobaric hypoxia on diaphragmatic myofilament contractile function and phosphorylation in high-altitude deer mice and low-altitude white-footed mice

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O₂ represents a challenge to maintaining oxygen delivery to tissues. Previous work has demonstrated that hypoxia acclimation of deer mice and low altitude white-footed mice (P. leucopus) increases the force generating capacity of the diaphragm. The mechanism behind this improved contractile function is not known. Within myocytes, the myofilament plays a critical role in setting the rate and level of force production, and its ability to generate force can change in response to changes in physiological conditions. In the current study, we examined how chronic hypobaric hypoxia exposure of deer mice and white-footed mice influences the Ca²⁺ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca²⁺ sensitivity of force generation, were not impacted by acclimation to hypobaric hypoxia, and did not differ between preparations from the two species. The cooperativity of the force-pCa relationship, and the maximal rate of force generation were also the same in the preparations from both species, and not impacted by acclimation. Finally, the relative phosphorylation of TnT, and MLC was lower in deer mice than white-footed mice, but was not affected by acclimation. These results indicate that species differences in diaphragm function, and the increase in force production with hypoxia acclimation, are not due to differences, or changes, in myofilament function. However, it appears that diaphragmatic myofilament function in these species is not affected by chronic hypobaric hypoxia exposure.

     

Sodium-Calcium Exchange Is Essential for Effective Triggering of Calcium Release in Mouse Heart

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca²⁺ release triggered by Ca²⁺ influx through L-type Ca²⁺ channels. Although Na⁺-Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca²⁺ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca²⁺ and normal Ca²⁺ transients despite reduced L-type Ca²⁺ current. We loaded myocytes with fluo-3 to image Ca²⁺ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca²⁺ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca²⁺, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca²⁺ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca²⁺ release was asynchronous. These results suggest that high subsarcolemmal Ca²⁺ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca²⁺ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca²⁺ for normal triggering, even when subsarcolemmal Ca²⁺ is lowered by EGTA. Thus, reducing subsarcolemmal Ca²⁺ with EGTA in NCX KO mice reveals the dependence of Ca²⁺ release on NCX.     

Passive Ca binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca²⁺ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca²⁺ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca²⁺ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca²⁺] ([Ca²⁺]_F was measured with Indo-1 and bound Ca²⁺ ([Ca²⁺]_B) was the difference between [Ca²⁺]_F and total Ca²⁺. Apparent Ca²⁺ dissociation constants (K_d) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl₂ additions (2.5–10 nmoles) yielded δ[Ca²⁺]_B/δ[Ca²⁺]_F for the sum of [Ca²⁺]_B at all three classes of binding sites. From this function, the apparent number of endogenous sites (B_en) and their K_d (K_en) were determined. Similar K_en values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, B_en was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that B_en determined in VH underestimates cellular B_en by 29%. Although B_en values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing B_en per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca²⁺ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca²⁺ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.     

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)     

Activated expression of cardiac adenylyl cyclase 6 reduces dilation and dysfunction of the pressure-overloaded heart

Background and objective

Cardiac-directed adenylyl cyclase 6 (AC6) expression attenuates left ventricular (LV) hypertrophy and dysfunction in cardiomyopathy, but its effects in the pressure-overloaded heart are unknown.

Methods

Mice with cardiac-directed and regulated expression of AC6 underwent transaortic constriction (TAC) to induce LV pressure overload. Ten days prior to TAC, and for the duration of the 4 week study, cardiac myocyte AC6 expression was activated in one group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 4 week after TAC, and LV samples assessed for alterations in Ca²⁺ signaling.

Results

LV contractility, as reflected in the end-systolic pressure–volume relationship (E_max), was increased (p = 0.01) by activation of AC6 expression. In addition, diastolic function was improved (p < 0.05) and LV dilation was reduced (p < 0.05). LV samples from AC-On mice showed reduced protein expression of sodium/calcium exchanger (NCX1) (p < 0.05), protein phosphatase 1 (PP1) (p < 0.01), and increased phosphorylation of phospholamban (PLN) at Ser16 (p < 0.05). Finally, sarcoplasmic reticulum (SR) Ca²⁺ content was increased in cardiac myocytes isolated from AC-On mice (p < 0.05).

Conclusions

Activation of cardiac AC6 expression improves function of the pressure-overloaded and failing heart. The predominant mechanism for this favorable adaptation is improved Ca²⁺ handling, a consequence of increased PLN phosphorylation, reduced NCX1, reduced PP1 expression, and increased SR Ca²⁺ content.