Changes In Calcium Sensitivity Research Articles

Regulation of vascular smooth muscle cell contraction during early postnatal ontogenesis

Growth of the body in early postnatal ontogenesis is associated with changes in the functioning of many organ systems, including the cardiovascular system. The circulatory system of newborns is characterized by numerous structural and functional features, which at the systemic level is manifested in a significantly lower level of blood pressure. This review describes the differences in the mechanisms of regulation of vascular smooth muscle cell contraction in early postnatal ontogenesis and in adulthood, including age-related changes in the functioning of ion channels, which activity affects membrane potential level and intracellular concentration of calcium ions, as well as changes in calcium sensitivity of the contractile apparatus. The final section of the review discusses the connection between the mechanisms regulating contraction and differentiation of vascular smooth muscle cells during maturation.

Abstract Mo120: Overexpression of Prostaglandin E2 EP3 Receptor Subtype Alters Calcium-Handling Proteins in Mouse Hearts

Prostaglandin E2 (PGE2) is an autacoid that acts through G-protein coupled receptors (EP1-EP4) in various cell types within the heart, including cardiomyocytes in which EP3 and EP4 predominate. We previously reported that mice with cardiomyocyte-specific overexpression of the EP3 receptor (EP3 TG) demonstrate reduced cardiac function and diminished cardiomyocyte contractility in isolated cardiomyocytes. We therefore hypothesized that PGE2 via the EP3 receptor regulates calcium handling proteins to reduce contractility. To test our hypothesis, we performed RNA sequencing (RNA seq) on the left ventricle of fifteen-week-old male EP3 TG mice along with their WT controls. Western blots were used to confirm changes in the expression of calcium-handling proteins such as Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Ryanodine receptor 2 (RYR2), Phospholamban (PLB), Troponins (C, I, T) and voltage-gated calcium channel (CACNB2). For RNA seq, all data is reported as Log2Fold change, adjusted P value, and western blot data is reported as mean ± standard error (SE) of the mean. Additionally, isolated cardiomyocyte contractility was assessed using the IonOptix system, and data is reported as the mean ± SE. RNA seq data shows that SERCA2a and RYR2 expression were significantly downregulated in TG mice (SERCA2a -0.44, p=4.72 x 10 -6 ; RYR2: -0.86, p=4.08 x 10 -5 ) in addition to significant reductions in Troponin I (-0.43, p=0.038) and the Voltage-gated calcium channel (CACNB2: -0.71,p=0.0055). Western blot analyses confirmed the reductions in SERCA2a, RYR2, and CACNB2 although these changes were only significant for CACNB2 (1.0 ± 0.03 for WT vs 0.59 ± 0.04 for TG, p = 0.016). Expression of Troponins did not change in TG mice. Consistent with our previous data, IonOptix analysis shows reduced contractility of myocytes from EP3 TG mice (bl% peak h is 3.47 ± 0.20 for WT vs 1.0 ± 0.14 for TG, p<0.0001) despite a similar increase in the Ca 2+ transients (Ca 2+ peak h for WT= 0.86 ± 0.04 and TG=0.86 ± 0.07) suggesting potential changes in the sensitivity of the myofilament to Ca 2+ . In conclusion, the reduced contractility observed in EP3 TG mice may be due to alterations in calcium handling proteins and/or changes in calcium sensitivity at the level of the myofilament.

Tropomyosin R160A is crucial for TnI phosphorylation-induced effects on calcium sensitivity.

The thin filament, consisting of the three-protein complex troponin (TnI, TnT, and TnC) and tropomyosin (Tpm) bound to filamentous actin, acts as the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, TnI biases Tpm in a position on actin that blocks myosin binding whereas calcium binding to TnC causes a propagated conformational change in Tn/Tpm that relieves this inhibition. During heart failure and adrenergic stimulation, phosphorylation of the N-terminal region of troponin I (TnI) at residues S23 and S24, has been shown to play a role in heart muscle adaptation through a decrease in calcium sensitivity. Previous modeling studies performed in thin filament models have suggested that positively charged groups on Tpm, namely R160, could interact with the phosphorylated serine residues on TnI. Here we tested the hypothesis that interactions between Tpm-R160 and phosphorylated S23/24 participate in phosphorylation-induced effects on calcium sensitivity (Pavadai et al., 2022). Tpm mutated from wild-type R160 to alanine (Tpm-R160A) and phosphomimetic TnI affecting both serine 23 and 24 (S23/24D) were generated and expressed recombinantly. Using in vitro motility (IVM) assays, we quantified the effects of these mutations alone and in combination. Regulated IVM experiments using thin filaments reconstituted with phosphomimetic S23/24D-TnI and wild-type Tpm revealed a decrease in calcium sensitivity, showing that phosphorylation is successfully mimicked by mutating serine to an aspartic acid,consistent with previous studies on human cardiac tissue. However, when TnI-S23/24D is paired with mutant R160A-Tpm, the phosphorylation-induced changes in calcium sensitivity are diminished. These results are consistent with Tpm-R160 participating in the lusitropy observed when the N-terminal of TnI is phosphorylated.

A Discrete Loop of the SERCA N-Domain Interacts with Phospholamban and Stabilizes a Compact Conformation of the SERCA Cytosolic Headpiece

The cardiac sarco/endoplasmic reticulum (ER) calcium ATPase (SERCA) and its inhibitor phospholamban (PLB) are key regulators of calcium levels in cardiac cells. In previous molecular dynamics (MD) simulations of SERCA spontaneous headpiece closure, we identified a discrete N-domain beta 5,6-loop that directs the closure of SERCA headpiece. Here we use additional MD simulations to pinpoint specific negative residues that facilitate SERCA headpiece closure. In addition, fluorescence resonance energy transfer (FRET) measurements showed that mutation of these key residues slows down calcium-dependent SERCA headpiece closure, as predicted from MD calculations. The functional significance of impaired SERCA headpiece closure was demonstrated by a 60% decrease in SERCA ATPase activity with no detectable change in calcium sensitivity. Additionally, loop mutations reduced transport of calcium as measured by a low affinity calcium sensor (R-CEPIA1er) in the ER of HEK cells. Interestingly, MD simulations of the SERCA-PLB regulatory complex model showed that PLB phosphorylation on Serine 16 stabilized a compact, ordered SERCA headpiece through an interaction with the beta 5,6-loop. Fluorescence lifetime imaging microscopy (FLIM) measurements also detected a conformational change of the SERCA-PLB regulatory complex after phosphomimetic mutation of PLB. Overall, the data suggest that this discrete loop is important for SERCA transport activity and functional regulation by PLB. This novel structural element may represent a target for modulation of SERCA activity.

Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I.

Control of calcium binding to and dissociation from cardiac troponin C (TnC) is essential to healthy cardiac muscle contraction/relaxation. There are numerous aberrant post-translational modifications and mutations within a plethora of contractile, and even non-contractile, proteins that appear to imbalance this delicate relationship. The direction and extent of the resulting change in calcium sensitivity is thought to drive the heart toward one type of disease or another. There are a number of molecular mechanisms that may be responsible for the altered calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC. Based on our previous work, TnI peptide binding studies and the calcium binding properties of chimeric TnC-TnI fusion constructs, and building upon the concept of effective concentration, we have developed a mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We predict that several TnI and TnT modifications do not alter any of the intrinsic calcium or TnI binding constants of TnC, but rather alter the ability of TnC to “find” TnI in the presence of calcium. These studies demonstrate the apparent consequences of the effective TnI concentration in modulating the calcium binding properties of TnC.

Effect of Amino Acid Changes in a Troponin I FHC Hotspot on Protein:Protein Binding and Calcium Sensitivity of Force Development

Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI that is associated with familial hypertrophic cardiomyopathy (FHC) is arginine, which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. To determine the effect of these mutations (R204C and R204H), as well as other cTnI mutations, R204P, R204Q, and R204W, calcium-force measurements and cTnI:troponin C (TnC) and cTnI:troponin T (TnT) interactions using the mammalian two-hybrid luciferase assays were utilized. All five mutations showed significant increases in calcium sensitivity of force development ranging from ΔpCa50 0.23 (R204W) to 0.35 (R204P). The mutations associated with FHC, R204C and R204H, had ΔpCa50 values of 0.28 and 0.29 respectively. The cTnI containing the R204P mutation showed the weakest interaction with TnT when compared to wild-type cTnI or the other mutants. The R204H mutation also showed significant impairment in its ability to interact with TnT, while the R204C mutation showed mild impairment when compared to wild-type cTnI. The R204C and R204P mutations showed the greatest impairment in binding to TnC. These results suggest that mutations at the same site on cTnI could affect thin filament interactions differentially, and that significant impairment in the interaction of cTnI with TnT or TnC may be enough to cause significant changes in calcium sensitivity. If the large increase in calcium sensitivity of force development observed with these mutations is associated with the poor prognosis then other R204 mutations are likely to have a poor prognosis. This research was supported by a Hellman Fellowship.

Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca2+-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca2+ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca2+ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.

Abstract 17270: The Effects of Cardiac Resynchronization Therapy on Myofilament Calcium Sensitivity are Global and Mediated by Protein Phosphorylation

We have previously shown that calcium sensitivity is significantly decreased with dyssynchrony, but is improved with cardiac resynchronization therapy (CRT) using trabeculae from the right ventricle (RV). However, dyssynchrony and resynchronization cause regional changes in stress, with the RV and left ventricle (LV) experiencing differing loads. To investigate the regional effects of CRT, we used 3 canine models: dyssynchronous heart failure (HF dys , left bundle ablation, 6 wks atrial tachypacing), CRT (left bundle ablation, 3 wks atrial tachypacing, 3 wks biventricular tachypacing), and non-paced controls. Steady-state force-calcium relationships were measured in skinned myocytes isolated from the LV lateral wall. Compared to control, HF dys exhibited decreased maximal calcium-activated force (F max , Con: 35.00 ± 4.61 93 mN•mm -2 , n = 7 myocytes from 3 dogs, HF dys : 13.44 ± 1.66 mN•mm -2 , n = 7 myocytes from 3 dogs, P < 0.01), and decreased calcium sensitivity (EC 50 , Con: 1.92 ± 0.16 μM, HF dys : 2.58 ± 0.11, P < 0.01). These parameters were returned to near control values in the CRT dog (F max : 36.16 ± 2.78 mN•mm -2 , EC 50 : 2.00 ± 0.19 μM, n = 8 myocytes P < 0.05 vs HF dys , P = N.S. vs Con). To discover if these changes were mediated by altered phosphorylation state, we treated skinned RV trabeculae muscles with the phosphatase PP1 (2,500 U/ml) for 60 minutes, and measured force-calcium curves before and after treatment. None of the groups had a statistically significant change in F max after treatment ( P = N.S.). However, there was an increase in EC 50 in both Con (ΔEC 50 : 1.09 ± 0.22 μM, n = 6) and CRT (ΔEC 50 : 0.70 ± 0.23 μM, n = 5), while there was no effect on HF dys trabeculae (ΔEC 50 : -0.06 ± 0.14 μM, n = 5, P < 0.05 vs Con and CRT). This suggests that myofilament protein phosphorylation is responsible for the changes in calcium sensitivity. In summary, these results show that despite opposing regional changes in stress, the myofilament responds in a globally homogenous manner and is mediated by altered global kinase/phosphatase activity, and not by local stress response. Overall, this is encouraging data for the development of a pharmacological intervention to recreate the beneficial aspects of CRT, since regional targeting may be unnecessary.

The Effect of Long-term Hypoxia on Tension and Intracellular Calcium Responses Following Stimulation of the Thromboxane A2 Receptor in the Left Anterior Descending Coronary Artery of Fetal Sheep

The purpose of this study was to investigate the mechanisms of tension and intracellular calcium regulation following stimulation with the thromboxane A(2) receptor agonist U46619 in the left anterior descending coronary artery of fetal sheep exposed to long-term hypoxia. We hypothesized that there would be a reduction in intracellular calcium responses in long-term hypoxic left anterior descending coronary artery accompanied by an increase in calcium sensitivity of the contractile mechanism. Pregnant sheep were kept at altitude (3820 m) from day 30 of gestation until day 140. Fetal hearts from long-term hypoxic and from a control, normoxic group were obtained and the left anterior descending coronary artery of the fetus was dissected, cleaned, and mounted in a bath (Jasco) in which tension and intracellular calcium [Ca(2+)](i), using Fura-2, could be measured simultaneously following stimulation of the thromboxane A(2) receptor with U46619. The role of intracellular calcium and the Rho kinase and protein kinase C pathways in the tension responses were investigated by maintaining intracellular calcium constant or by using the Rho kinase blocker, Y27632, or the protein kinase C blocker, GF109203-X. There was no difference in the tension dose-response to U46619 between the normoxic fetal and hypoxic fetal left anterior descending, although [Ca(2+)](i) was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca(2+)]( i) was maintained constant at baseline levels, U46619 produced the same tension dose-response in both normoxic fetal and hypoxic fetal left anterior descending as when [Ca(2+)](i) was allowed to rise. The tension response was abolished in both groups when the Rho kinase inhibitor, Y27632, was given either during or before stimulation with U46619. The protein kinase C blocker, GF109203-X, had no effect on the tension response in either group. Long-term hypoxia did not alter the tension response to thromboxane A(2) receptor stimulation in fetal left anterior descending. The contractions in response to U46619 were produced apparently completely by changes in calcium sensitivity through the Rho kinase pathway.

Abstract 5403: Protein Kinase C-Induced Troponin I Phosphorylation Causes Changes in Cardiac Contractile Function but not the Intracellular Calcium Transient

Both intracellular calcium transients ([Ca] i ) and myofilament properties determine cardiac muscle contractile force. Transgenic mouse models created to perturb specific myofilament proteins often cause a compensatory change in [Ca] i , which confounds the assessment of myofilament structure-function relationships. We have created a new transgenic mouse that has all three protein kinase C (PKC) phosphorylation sites on cardiac troponin I (cTnI) mutated to glutamic acid, rendering these sites constitutively pseudo-phosphorylated. Our goal was to determine the effects of this mutation on cardiac muscle contractile function and whether these effects would be concurrent with changes in the [Ca] i . Two sets of studies were conducted: skinned muscle fiber experiments to characterize the steady-state force-pCa relationships at sarcomere lengths of 1.9 and 2.3 μm and right ventricular papillary muscle experiments to characterize the peak developed force (F dev )-muscle length (L) relationships and [Ca] i (fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm, R = [emission fluorescence 340 ]/[emission fluorescence 380 ]). In skinned fibers, there was a significant decrease in maximally activated force (i.e., force at pCa 4.33) in transgenic mice (Wild-Type, WT (n = 7): 64.4± 8.0, Transgenic, TG (n = 6): 42.6±6.8 mN•mm −2 , P = 0.004), without any changes in calcium sensitivity or cooperativity (Hill coefficient). In intact papillary muscles, TG mice showed a decrease in F dev and slowed relaxation for all muscle lengths examined (F dev @ 100% L max , WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm −2 , P = 0.005; dF/dt min @ 100% L max , WT: −136±32, TG: −74±38 mN•mm −2 •s −1 , P = 0.002). In contrast, [Ca] i was unaltered in TG mice at all muscle lengths examined ([Ca] i amplitude as quantified by R systole / R diaastole , WT: 1.62±0.07, TG: 1.48±0.22; [Ca] i relaxation rate d R /dt min , WT: −96±37, TG: −64±30 s −1 ). Thus, PKC-induced TnI phosphorylation affects cardiac muscle contraction (reduced force magnitude and slowed relaxation) via changes in the myofilament properties (activation and/or crossbridge dynamics), and these contractile effects are not related to any changes in the intracellular calcium transient.

Calcium‐dependent protein kinase C alpha and the frequency‐dependent increase in phosphorylation of troponin I in failing hearts

In healthy myocardium the force‐frequency relation, one of the key factors modulating cardiac performance, is positive, while in failing myocardium a negative relation is found. This difference has been ascribed to changes in calcium homeostasis and, more recently, to changes in calcium sensitivity through increased phosphorylation of troponin I (TnI). Our objective was to determine whether altered activation of calcium‐dependent protein kinase C alpha (PKCα) determines the increase in phosphorylation of TnI with frequency in failing hearts.Right ventricular heart failure was induced by monocrotaline (MCT; 80mg/kg). After 4 weeks isovolumic pressure–frequency relations were determined in Langendorff‐perfused hearts in a control (n=19) and MCT‐treated group (n=19). Thereafter, hearts were kept quiescent or paced at 9 Hz for 60 min and flash frozen to determine phosphorylation of TnI and PKCα by 1D‐gel electrophoresis.The pressure–frequency relation was constant in control and negative in MCT group. Phosphorylation of TnI did not depend on frequency in control but increased in MCT group. Protein expression and phosphorylation levels of PKCα were both increased with frequency in MCT group.These results indicate that frequency‐dependent changes in PKC‐phosphorylatable sites of TnI in failing hearts originate from an altered balance in calcium‐dependent kinase/phosphatase activity.

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

Age‐Dependent Changes in the Calcium Sensitivity of Striatal Mitochondria in Mouse Models of Huntington's Disease

Striatal and cortical mitochondria from knock-in and transgenic mutant huntingtin mice were examined for their sensitivity to calcium induction of the permeability transition, a cause of mitochondrial depolarization and ATP loss. The permeability transition has been suggested to contribute to cell death in Huntington's Disease. Mitochondria were examined from slowly progressing knock-in mouse models with different length polyglutarnine expansions (Q20, Q50, Q92, Q111) and from the rapidly progressing transgenic R6/2 mice overexpressing exon I of human huntingtin with more than 110 polyglutamines. As previously observed in rats, striatal mitochondria from background strain CD1 and C57BL/6 control mice were more sensitive to calcium than cortical mitochondria. Between 5 and 12 months in knock-in Q92 mice and between 8 and 12 weeks in knock-in Q111 mice, striatal mitochondria developed resistance, becoming equally sensitive to calcium as cortical mitochondria, while those from Q50 mice were unchanged. Cortical mitochondrial calcium sensitivity did not change. In R6/2 mice striatal and cortical mitochondria were equally resistant to Ca2+ while striatal mitochondria from littermate controls were more susceptible. No increases in calcium sensitivity were observed in the mitochondria from Huntington's Disease (HD) mice compared to controls. Neither motor abnormalities, nor expression of cyclophilin D corresponded to the changes in mitochondrial sensitivity. Polyglutamine expansions in huntingtin produced an early increased resistance to calcium in striatal mitochondria suggesting mitochondria undergo compensatory changes in calcium sensitivity in response to the many cellular changes wrought by polyglutamine expansion.

Age effects on the adaptive response of the female rat heart following aortic constriction.

Aging is associated with adaptations in the hearts of mammals that diminish the reserve capacity to meet hemodynamic loading challenges. To evaluate potential mechanisms of this phenomenon, the following hypotheses were tested: compared with hearts of adult rats, hearts of aged rats undergoing aortic constriction will exhibit (a) a lower concentration of myofibrillar proteins, (b) a reduced sensitivity to extracellular calcium, and (c) a reduced coronary perfusion. Female Fischer 344 rats aged 9 months (adult) and 27 months (aged) were assigned to control (C) or aortic-constriction (AC) groups and studied at 7 and 28 days post-AC, yielding six groups of rats. Analysis of variance was used to examine the effects of age and AC. The left ventricular (LV) mass/body mass ratio expressed a percentage of age-matched control value averaged AC-7adult, 111%; AC-28adult, 120%; AC-7aged, 106%; AC-28aged, 123% (AC, p < .01). As a percentage of adult rats values, the pressure-generating capacity of the LV averaged Caged, 99%; AC-7aged, 92%; AC-28aged, 92% (age, p < .05). There were no differences attributable to age or AC in either myofibrillar protein concentration or calcium sensitivity. There was, however, a significantly lower concentration of nonmyofibrillar protein (approximately 10%) in the hearts of all three groups of aged rats compared with the adult rats that was unaltered by AC. The percentages of LV myosin heavy chain in the alpha-isoform were Cadult, 77%; AC-7adult, 66%; AC-28adult, 66%; Caged, 45%; AC-7aged, 41%; AC-28aged, 32% (age, p < .01; AC,p < .01). Coronary flow per gram of tissue averaged 9% lower in all three of the aged groups compared with the adult rats and was not significantly affected by AC (age, p < .05). The data suggest that a reduction in nonmyofibrillar protein and a reduced coronary flow, rather than changes in calcium sensitivity or myofibrillar protein, are associated with an impairment in the adaptive response of the aged heart.

Analysis of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles using models derived from single-channel data

The planar lipid bilayer and vesicle release experiments are two alternative approaches used to study the function of the ryanodine receptor (RyR) channel at the subcellular level. In this work, we combine models of gating (Zahradnı́ková and Zahradnı́k, Biophys. J. 71 (1996) 2996–3012) and permeation (Tinker et al., J. Gen. Physiol. 100 (1992) 495–517) of the cardiac RyR channel to simulate calcium release experiments on sarcoplasmic reticulum vesicles. The resulting model and real experimental data agreed well within the experimental scatter, confirming indistinguishable properties of the RyRC in the vesicle preparation and in the planar lipid bilayer. The previously observed differences in calcium dependencies of the release and the gating processes can be explained by binding of calcium within the RyRC conducting pore. A novel method of analysis of calcium dependence of calcium release was developed and tested. Three gating models of the RyRC, showing, respectively, an increase, no change, and a decrease in calcium sensitivity over time, were compared. The described method of analysis enabled determination of temporal changes in calcium sensitivity, giving potential for detection of the adaptation/inactivation phenomena of the RyRC in both vesicle and in situ release experiments.

Anthracyclines enhance tension development in cardiac muscle by direct interaction with the contractile system.

Anthracyclines are highly effective anticancer agents which induce a well described but incompletely understood cardiac toxicity. In this study, a direct action of several anthracyclines on the force generating mechanism of heart muscle preparations is described. To allow discrimination between membrane related effects and a direct action of anthracyclines on the actin-myosin contractile system, both inner and outer membranes of cardiac fibres were permeabilized. All anthracyclines tested in this study [doxorubicin (Dox), epirubicin, daunorubicin and idarubicin] showed positive inotropic actions. Dox and epirubicin, which are considered the most cardiotoxic drugs of the anthracycline family, significantly increased the maximal calcium activated tension by 33% (n = 8, P < 0.01) and by 26% (n = 8, P < 0.01) respectively. Daunorubicin and idarubicin increased the maximal tension by 12% and 9% respectively (P = n.s.). Other chemotherapeutic drugs (Taxol and 5-FU) had no effect on maximal tension. To elucidate the mechanism behind this Dox-induced increase in maximal tension, calcium sensitivity curves were measured and rigor experiments were performed. A small but significant increase in pCa50 value (+0.14 +/- 0.03, P < 0.05) was observed only after incubation with 20 microM Dox. Dox acted during the transition to force generating cross-bridges as reflected by the significant increase in rigor tension (12%, P < 0.05) after preincubation of cardiac fibres with Dox. Cycling of cross-bridges is a prerequisite for Dox to increase tension because no effect on tension was seen after Dox was added to fibres in an established rigor. In summary, anthracyclines increased the maximal tension in cardiac muscle fibres by direct interaction with the actin-myosin cross-bridges. Changes in calcium sensitivity are unlikely to contribute to the observed increase in maximal tension. The rise in tension as is seen in this experimental set-up may contribute to destruction of the contractile machinery of cardiac muscle. In agreement with this hypothesis is the observation that the more cardiotoxic anthracyclines induced the largest increase in maximal tension of the cardiac fibres.

Altered contractile function in isoproterenol-induced hypertrophied rat heart.

To study the calcium-dependent mechanisms contributing to altered contractile function in isoproterenol-induced ventricular hypertrophy of the rat heart. The force-interval relationship, systemic evaluation of changed contractile force and calcium sensitivity of the myofilaments were investigated using small trabecular muscle from hearts with isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by daily subcutaneous injection of isoproterenol for 12 days. The calcium sensitivity of myofilaments was studied using non-ionic detergent (Triton-X-100)-skinned trabeculae. The contractile force was evaluated at various concentrations of extracellular calcium and muscle lengths. The force-interval relationship was used to reflect altered intracellular calcium handling. The isoproterenol-induced cardiac hypertrophy was associated with significantly enhanced contractile force at various concentrations of extracellular calcium and muscle lengths. Also, an amplified force-interval relationship in hypertrophied muscle at long rest intervals was found. However, this study revealed no change in calcium sensitivity of myofilaments. Altered intracellullar calcium handling contributes to enhanced contractile force in isoproterenol-induced cardiac hypertrophy.

Thiophosphorylation of the 130-kDa Subunit Is Associated with a Decreased Activity of Myosin Light Chain Phosphatase in α-Toxin-permeabilized Smooth Muscle

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

Chronic administration of nifedipine induces upregulation of dihydropyridine receptors in rabbit heart.

In rabbits chronically pretreated with nifedipine (20 mg/day for 25 days), we demonstrate upregulation of Ca2+ channels in mammalian heart (maximal binding capacity = 222 +/- 14 and 421 +/- 55 fmol/mg protein for control and treated, respectively; P < 0.05) without significant changes in dissociation constant. No changes in calcium sensitivity or maximal force were detected by pretreatment with nifedipine in chemically skinned fibers. Cardiac contractility at different extracellular Ca2+ concentrations was similar in control and pretreated animals. This upregulation of Ca2+ channels in mammalian heart offers an explanation for the lack of significant negative inotropic effect after chronic administration of nifedipine.

Influence of filling on left ventricular diastolic pressure-volume curve during pacing ischemia in dogs.

The reversible upward shift of the diastolic pressure-volume curve that occurs during pacing-induced ischemia has not been fully explained by increases in passive chamber stiffness or reductions in relaxation rate. We measured the fully relaxed pressure-volume relation defined by both filling and nonfilling beats and the isovolumic relaxation time constant in nonfilling beats before and during demand ischemia using our in situ left ventricular volume clamping technique in 10 dogs. Pacing-induced ischemia shifted the diastolic pressure-volume curves in filling beats upward compared with the end-diastolic pressure-volume relation of the normally perfused heart. In contrast, the end-diastolic points for nonfilling beats during pacing-induced ischemia fell on the fully relaxed pressure-volume relation defined by the normally perfused heart. Left ventricular filling per se was necessary for the upward shift of the diastolic pressure-volume curve observed during pacing-induced ischemia. We speculate that active force developed during diastole induced by stretch activation or, perhaps, length-dependent changes in calcium sensitivity of the myofilaments in the ischemic myocardium due to stretch of the myocardium during rapid diastolic filling may contribute to the upward shift of the diastolic pressure-volume curve observed during pacing-induced ischemia.