Altered RyR2 regulation by the calmodulin F90L mutation associated with idiopathic ventricular fibrillation and early sudden cardiac death

Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation–contraction coupling. Defective CaM–RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM^F90L) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM^F90L. F90L confers a deleterious effect on protein stability. Ca²⁺-binding studies reveal reduced Ca²⁺-binding affinity and a loss of co-operativity. Moreover, CaM^F90L displays reduced RyR2 interaction and defective modulation of [³H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca²⁺ release via aberrant CaM^F90L–RyR2 interaction is a potential mechanism that underlies familial IVF.     

Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

Previous reports indicate that reactive oxygen species (ROS) may modulate contractility in skeletal muscle. Although Ca(2+)-sensitivity of the contractile apparatus appears to be a primary site of regulation, dihydropyridine receptor (DHPR or L-type Ca(2+) channels) and calcium efflux in isolated sarcoplasmic reticulum (SR) vesicles appear to be redox sensitive as well. However, DHPR as a target is poorly understood in intact muscles at body temperature, particularly in the diaphragm, a muscle more dependent on external Ca(2+) than locomotor muscles. Previously, we reported that oxidant challenge via xanthine oxidase (XO) alters the K(+) contractures in diaphragm fiber bundles, suggestive of a role of L-type Ca(2+) channels. Contractility of isolated rat diaphragm fiber bundles revealed a biphasic response to ROS challenge that was dose and time dependent. Potentiation of twitch and low-frequency diaphragm fiber bundle contractility with 0.02 U?ml(-1) XO was reversible or partially preventable with washout, dithiothreitol, and the SOD/catalase mimetic EUK-134. The RyR antagonist ruthenium red inhibited xanthine oxidase-induced potentiation, while the RyR agonist caffeine elevated diaphragm twitch and low-frequency tension in a non-additive manner by 55% when introduced simultaneously with ROS challenge. The DHPR antagonist nitrendipine (15 μM) inhibited elevation in low-frequency diaphragm tension produced by ROS challenge. Caffeine threshold tension curves were shifted to the left with 0.02 U?ml(-1) XO, but this effect was partially reversed with 15 μM nitrendipine. These results are consistent with the hypothesis that DHPR redox state and RyR function are modulated in an interactive manner, affecting contractility in intact diaphragm fiber bundles.     

The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.     

Modeling atrial and ventricular contractility of the heart muscle. I. Modeling single contractions

A system of differential equations describing myocardium contractions in isometric and isotonic regimes has been obtained. On the basis of these equations functioning of myocardium was modelled on an electronic computer. An effect has been revealed of the coefficients of rheological equations and activation function on isometric and isotonic contraction of the force/rate ratio. A good agreement between the results of modelling and the experimental data has been observed.     

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca²⁺ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca²⁺ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca²⁺ activation and abolished luminal Ca²⁺ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [³H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.     

A centronuclear myopathy--dynamin 2 mutation impairs autophagy in mice

Dynamin 2 (Dnm2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous (HTZ) mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot-Marie-Tooth neuropathy. We generated a knock-in Dnm2R465W mouse model expressing the most frequent human mutation and recently reported that HTZ mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous (HMZ) mice. We show that HMZ mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in HMZ embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved HMZ cells have a higher number of immature autophagy-related structures probably due to a defect of acidification. Our results highlight the role of Dnm2 in the cross talk between endosomal and autophagic pathways and evidence a new role of Dnm2-dependent membrane trafficking in autophagy which may be relevant in DNM2-related human diseases.     

Cold adaptation suppresses the contractility of both atrial and ventricular muscle of the crucian carp heart

Adaptation to low temperature in crucian carp Carasius carassius increases twitch duration both in atrial and ventricular muscle and the response is largely explained by a temperatureinduced reduction in myofibrillar ATPase activity. The prolonged twitch increases the refractoriness of both atrial and ventricular muscle, i.e. the optimal force development is achieved at longer diastolic intervals after acclimation to cold. The contractions of atrial and ventricular muscle are insensitive to 10 μ ryanodine, an inhibitor of SR release Ca²+ channel but sensitive to 20 μ verapamil, a blocker of L-type Ca²+ channels. This suggests that E-C-coupling in the crucian carp heart is exclusively based on extracellular Ca²⁺ sources. The present findings indicate that cold-acclimation slows the contraction kinetics of the crucian carp heart and thereby preconditions the cardiac muscle for a low energy supply during winter anoxic conditions.     

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

In the thyroid gland Duox2-derived H₂O₂ is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2^thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H₂O₂, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.     

Correlation of atrial and ventricular contractility. The possibility of using the atria for controlling the artificial heart]

A possibility of the artificial ventricle regulation using information on the atrial contraction is discussed. Correlations between atrial wall tension and ventricle tension as well as the pressure under radom heart rhythm variations were studied. The wall tension of the appropriate heart portion was recorded by the arched tensiometers, and intraventricular pressure under cavity catheterization by the electric manometer. The cardiac rhythm varied between 2.0 c-1 and 4.0 c-1. A correlation between the tension and interpulse interval, (correlation coefficient being 0.62 +/- 0.05) was established. The close relationships "atrial wall tension-intramuscular pressure" were observed. For such relationships the correlation coefficient changed from 0.713 +/- 0,09 to 0,874 +/- 0.02 depending on the average value of interpulse interval. It is concluded that the information on the atrial contraction can be used for artificial heart regulation.     

Phosphorylation of RyR2 and shortening of RyR2 cluster spacing in spontaneously hypertensive rat with heart failure

As a critical step toward understanding the role of abnormal intracellular Ca(2+) release via the ryanodine receptor (RyR(2)) during the development of hypertension-induced cardiac hypertrophy and heart failure, this study examines two questions: 1) At what stage, if ever, in the development of hypertrophy and heart failure is RyR(2) hyperphosphorylated at Ser(2808)? 2) Does the spatial distribution of RyR(2) clusters change in failing hearts? Using a newly developed semiquantitative immunohistochemistry method and Western blotting, we measured phosphorylation of RyR(2) at Ser(2808) in the spontaneously hypertensive rat (SHR) at four distinct disease stages. A major finding is that hyperphosphorylation of RyR(2) at Ser(2808) occurred only at late-stage heart failure in SHR, but not in age-matched controls. Furthermore, the spacing between RyR(2) clusters was shortened in failing hearts, as predicted by quantitative model simulation to increase spontaneous Ca(2+) wave generation and arrhythmias.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

A GABAA receptor mutation linked to human epilepsy (gamma2R43Q) impairs cell surface expression of alphabetagamma receptors

A mutation in the gamma2 subunit of the gamma-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type alpha1beta2gamma2 and mutant alpha1beta2gamma2R43Q GABARs expressed in HEK 293 cells. The gamma2R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing alpha1beta2gamma2R43Q GABARs, the number of binding sites for [3H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface alpha1beta2gamma2R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing alpha1beta2gamma2R43Q GABARs confirmed that surface expression of the gamma2R43Q subunit was reduced. These data demonstrate that the gamma2R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the gamma2R43Q subunit have epilepsy.     

The R1441C mutation alters the folding properties of the ROC domain of LRRK2

LRRK2 is a 250 kDa multidomain protein, mutations in which cause familial Parkinson's disease. Previously, we have demonstrated that the R1441C mutation in the ROC domain decreases GTPase activity. Here we show that the R1441C alters the folding properties of the ROC domain, lowering its thermodynamic stability. Similar to small GTPases, binding of different guanosine nucleotides alters the stability of the ROC domain, suggesting that there is an alteration in conformation dependent on GDP or GTP occupying the active site. GTP/GDP bound state also alters the self-interaction of the ROC domain, accentuating the impact of the R1441C mutation on this property. These data suggest a mechanism whereby the R1441C mutation can reduce the GTPase activity of LRRK2, and highlights the possibility of targeting the stability of the ROC domain as a therapeutic avenue in LRRK2 disease.     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

Expressional changes of RyR1 and RyR2 in PC12 cells after induction of apoptosis

Ca2+ released from endoplasmic reticulum through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) can trigger apoptotic or necrotic pathways in cooperation with proapoptotic and/or prosurvival proteins, as those of Bcl-2 family. In such regulatory pathways expressional modulation of these Ca2+ transporters could also be expected. Therefore, our aim was to determine the expressional changes of RyR1 and RyR2 after experimental induction of apoptosis in PC12 cells. Our results showed significant decrease of RyR1 and RyR2 expressions, while caspase-3 and Bax expression significantly increased. We conclude that induction of apoptosis in PC12 cells could result in RyR expression down regulation.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Indinavir impairs protein synthesis and phosphorylations of MAPKs in mouse C2C12 myocytes

Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated C2C12 myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 microM) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the S6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of mTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor eIF4G-eIF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation.     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

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.     

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)