Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca²⁺ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca²⁺ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (I_Na/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013. © 2013 Wiley Periodicals, Inc.     

Unaltered ryanodine receptor protein levels in ischemic cardiomvopathy

Previous studies on sarcoplasmic reticulum calcium release channel (ryanodine receptor) demonstrated that protein levels are unchanged in myocardium from hearts with end-stage failing dilated cardiomyopathy. In ischemic cardiomyopathy, ryanodine receptor mRNA levels were shown to be decreased but no data on protein levels are available. Accordingly, protein levels of ryanodine receptor, calsequestrin, and sarcoplasmic reticulum calcium-ATPase (SR-Ca²⁺-ATPase) were measured by Western blot analysis in nonfailing human myocardium (n = 7) and in end-stage failing myocardium due to ischemic cardiomyopathy (n = 14). Protein levels of calsequestrin which is the major sarcoplasmic reticulum calcium storage protein were similar in nonfailing myocardium and in myocardium from end-stage failing hearts with ischemic cardiomyopathy. Ryanodine receptor protein levels, normalized to total protein or calsequestrin were also unchanged in ischemic cardiomyopathy. In contrast, protein levels of SR-Ca²⁺-ATPase normalized to total protein or calsequestrin were decreased by 31 and 30%, respectively (p < 0.05). The data indicate that (I) sarcoplasmic reticulum calcium uptake sites are decreased relative to the release sites in ischemic cardiomyopathy, and (2) alterations of sarcoplasmic proteins are similar in ischemic and dilated cardiomyopathy.     

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

Abnormal cellular Ca²⁺ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca²⁺ transient amplitude due to decreased sarcoplasmic reticulum Ca²⁺ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca²⁺ release events which occur when sarcoplasmic reticulum Ca²⁺ content exceeds a certain threshold level. Such Ca²⁺ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca²⁺ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca²⁺ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca²⁺ transients and Ca²⁺ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca²⁺ content, decreased Ca²⁺ transient amplitude, and increased frequency of Ca²⁺ waves. These results are in line with a decreased threshold for diastolic Ca²⁺ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca²⁺ release were increased THR286 phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca²⁺ content and increased propensity for diastolic Ca²⁺ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca²⁺ handling after a myocardial infarction.     

Importance of the sarcoplasmic reticulum and adrenergic stimulation on the cardiac contractility of the neotropical teleost <Emphasis Type="Italic">Synbranchus marmoratus</Emphasis> under different thermal conditions

Experiments were carried out to investigate the heart rate of Synbranchus marmoratus after changing the temperature of the water contained in the experimental chamber of the acclimated fish (from 25 to 35°C and from 25 to 15°C). Then, an isometric cardiac muscle preparation was used to test the relative importance of Ca²⁺ released from the sarcoplasmic reticulum and Ca²⁺ influx across the sarcolemma for the cardiac performance under different thermal conditions: 25°C (acclimation temperature), 15 and 35°C. Adrenaline and ryanodine were used to modulate the Ca²⁺ flux through the sarcolemma and the sarcoplasmic reticulum, respectively. Ryanodine reduced the peak tension by approximately 47% at 25°C, and by 53% at 35°C; however, it had no effect at 15°C. A high adrenaline concentration was able to ameliorate the negative effects of ryanodine. Despite increasing the peak tension, adrenaline increased the times necessary for contraction and relaxation. We conclude that the sarcoplasmic reticulum is active in contributing Ca²⁺ to the development of tension at physiological contraction frequencies. The adrenaline-stimulated Ca²⁺ influx is able to increase the peak tension, even after addition of ryanodine, at physiologically relevant temperatures and pacing frequencies.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.     

Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts (n = 10) was performed using oxygenated Tyrode's solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl? (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (-17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (-26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (-11.3 ± 9.6%, P < 0.01) and its synchronization (-37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.     

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

The time-course of Ca²⁺ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca²⁺ indicator. Several methods were used to trigger Ca²⁺ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca²⁺ concentration ([Ca₀²⁺]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca²⁺ release induced by either halothane or Ca²⁺ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca²⁺ released by halothane at low [Ca₀²⁺] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca²⁺ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca²⁺ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum ($\text{k = 83}\text{s}{}^{\mathrm{?1}}$) than in normal ($\text{k = 37}\text{s}{}^{\mathrm{?1}}$). Thus, all types of Ca²⁺ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca²⁺ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.     

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca<Superscript>2+</Superscript>-channels and ryanodine receptors

L-type Ca²⁺-channel blockers, verapamil (5 μM) and nifedipine (10 μM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca²⁺-channel activator BAY K 8644 (1 μM) has a decreased mediator secretion level. Nifedipine (10 μM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 μM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 μM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 μM).     

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2–based ratiometric Ca²⁺-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca²⁺ imaging protocols revealed that HiK⁺ (30 mM)-induced intracellular free Ca²⁺ ([Ca²⁺]_i) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca²⁺]_i. This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca²⁺, providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.     

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: a possible role in myocardial ischemia.

Palmitylcarnitine is a time-dependent inhibitor of the Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.     

Beyond Bowditch: the convergence of cardiac chronotropy and inotropy

The ability of the heart to acutely beat faster and stronger is central to the vertebrate survival instinct. Released neurotransmitters, norepinephrine and epinephrine, bind to beta-adrenergic receptors (beta-AR) on pacemaker cells comprising the sinoatrial node, and to beta-AR on ventricular myocytes to modulate cellular mechanisms that govern the frequency and amplitude, respectively, of the duty cycles of these cells. While a role for sarcoplasmic reticulum Ca(2+) cycling via SERCA2 and ryanodine receptors (RyR) has long been appreciated with respect to cardiac inotropy, recent evidence also implicates Ca(2+) cycling with respect to chronotropy. In spontaneously beating primary sinoatrial nodal pacemaker cells, RyR Ca(2+) releases occurring during diastolic depolarization activate the Na(+)-Ca(2+) exchanger (NCX) to produce an inward current that enhances their diastolic depolarization rate, and thus increases their beating rate. beta-AR stimulation synchronizes RyR activation and Ca(2+) release to effect an increased beating rate in pacemaker cells and contraction amplitude in myocytes: in pacemaker cells, the beta-AR stimulation synchronization of RyR activation occurs during the diastolic depolarization, and augments the NCX inward current; in ventricular myocytes, beta-AR stimulation synchronizes the openings of unitary L-type Ca(2+) channel activation following the action potential, and also synchronizes RyR Ca(2+) releases following depolarization, and in the absence of depolarization, both leading to the generation of a global cytosolic Ca(i) transient of increased amplitude and accelerated kinetics. Thus, beta-AR stimulation induced synchronization of RyR activation (recruitment of additional RyRs to fire) and of the ensuing Ca(2+) release cause the heart to beat both stronger and faster, and is thus, a common mechanism that links both the maximum achievable cardiac inotropy and chronotropy.     

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

Internal calcium stores and norepinephrine overflow from isolated,field stimulated rat VAS deferens

Ryanodine has been shown to selectively inhibit the initial phase of contraction of rat vas deferens smooth muscle stimulated by endogenous release of norepinephrine (NE) (1), and part of this effect could be pre-junctional. To assess this, its effect on NE overflow was measured in the same preparation. NE overflow from electrical field-stimulated isolated rat vas deferens was quantified by electrochemical detection using HPLC. In order to limit pre-junctional autoregulatory mechanisms, α₂-adrenergic receptors were blocked and P_2x purinergic receptors were desensitized. In these experimental conditions, NE overflow was directly proportional to extracellular Ca²⁺ concentration. Ryanodine only induced a modest decrease in NE overflow. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase, slightly increased NE overflow but decreased smooth muscle contraction induced by electrical field stimulation. It is concluded that part of the effect of ryanodine on field stimulation-induced contraction may be due to an inhibition of NE release, although the major inhibitory effect of this alkaloid is post- junctional. For CPA, its inhibitory effect on field stimulation-induced contraction is entirely post-junctional. Its effect on NE overflow suggests that, in this preparation, internal Ca²⁺ stores could function to accelerate termination of neurotransmitter release by sequestering cytosolic Ca²⁺.     

Excitation-contraction coupling in heart muscle

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (I_ca) and [Ca²⁺]_i (with fura-2). We find that the voltage-dependence of Ica and the [Ca ²⁺]_i-transient and the dependence of [Ca²⁺]_i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (CICR) mechanism. Additionally, we find that when [Ca²⁺]_i and [Na⁺]_i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca²⁺i. Finally, measuring I_ca in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on I_ca that is independent of [Ca²⁺]_i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.