Effects of ryanodine on intracellular Ca2+ transients in mammalian cardiac muscle

We observed the effects of ryanodine on the aequorin luminescence, membrane potential, and contraction of canine cardiac Purkinje fibers and ferret ventricular muscle. In canine Purkinje fibers, ryanodine (10 nM to 1 microM) abolished the spontaneous spatiotemporal fluctuations in [Ca2+] that occur as a result of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) during exposure to low-Na+ solutions. Ryanodine strongly reduced the twitch and both components of the intracellular aequorin luminescence signal (L1 and L2), which normally accompanies contraction. The small luminescence signals that remained in ryanodine could be abolished by a Ca2+ channel blocker (nitrendipine, 10 microM). The plateau phase of the action potential was reduced by nitrendipine in the presence of ryanodine, which suggests that Ca2+ current was not blocked by ryanodine. In ferret ventricular tissue, ryanodine (1 microM) prolonged the action potential and reduced the peak amplitudes of both the aequorin transient and the twitch, while greatly prolonging the time-to-peak of both signals. Increases in extracellular [Ca2+] restored the peak amplitudes of the twitch and the aequorin luminescence, but did not restore the normal time-to-peak. The results show that in both tissues, the negative inotropic effect of ryanodine is due to the reduction of the intracellular [Ca2+] transient. Inasmuch as neither Ca2+ entry via surface membrane Ca2+ channels nor Na+-Ca2+ exchange appears to be blocked by ryanodine, the most probable cause of reduction of the [Ca2+] transient is an inhibition of Ca2+ release by the SR.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Decreased Ca2+ extrusion via Na+/Ca2+ exchange in epicardial left ventricular myocytes during compensated hypertrophy

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes

Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine- induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine- triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine- induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)- Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.     

Acetaldehyde depresses myocardial contraction and cardiac myocyte shortening in spontaneously hypertensive rats: role of intracellular Ca2+.

Acetaldehyde (ACA), the major metabolite of ethanol, exerts both stimulatory and depressive actions on myocardial tissue. We have recently shown that ACA depresses myocardial contraction, cardiac myocyte shortening and intracellular Ca2+ transients in normal rat heart. The purpose of the present study was to determine the influence of hypertension on ACA-induced myocardial actions. Mechanical properties of left ventricular papillary muscles and ventricular myocytes isolated from both 25-week-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were evaluated using force-transducer and video edge-detection, respectively. Papillary muscles and cardiac myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include: peak tension development (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT/+/-dL/dt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity (FFI) changes. ACA (1-30 mM) depressed PTD without affecting other mechanical indices in both WKY and SHR myocardium, with maximal inhibition of 64 and 69%, respectively. SHR myocytes exhibited increased cell dimension, baseline PTA and resting intracellular Ca2+ levels, compared to WKY counterparts. ACA (0.03-30 mM) depressed PTA without affecting TPT, TR90 and +/-dL/dt. The maximal inhibitions were 31 and 36% in WKY and SHR groups, respectively. Interestingly, ACA exerted a biphasic effect on FFI, displaying potentiation at lower doses (<3 mM) and inhibition at higher doses (>3 mM). The maximal increase in FFI changes were 19 and 22% at 0.3 mM and the maximal decreases were 37 and 29% at 30 mM ACA, in WKY and SHR myocytes, respectively. Neither resting intracellular Ca2+ levels (FFI) nor fluorescence decay time (FDT) were affected by ACA. The increase in FFI was attenuated by propranolol (1 microM), whereas the decrease in FFI was reversed by BayK 8644 (1 microM). These results suggest that hypertension does not appear to alter ACA-induced myocardial depression. The mechanism underlying ACA-induced myocardial actions may involve increased beta-adrenergic activity at low doses and reduced Ca2+ entry and/or release at high doses.     

Changes in mitochondrial functionality and calcium uptake in hypertensive rats as a function of age

We studied whether mitochondrial functions and Ca2+ metabolism were altered in Wistar Kyoto normotensive (WKY) and spontaneous hypertensive rats (SHR). Ca2+ uptake was decreased in SHR compared to WKY rats. Accumulation of Ca2+ was more efficient in WKY than in SHR rats. mDeltaPsi was lower in SHR compared to WKY rats. Basal complex IV activity was higher in SHR than WKY rats, whereas basal L-citrulline production, an indicator of nitric oxide synthesis, was decreased in SHR and dependent on Ca2+ concentration (p<0.05). Impact of Ca2+ was counteracted by EGTA. These data show an age-dependent decreased mitochondrial functions in brain mitochondria during hypertension.     

Time- and use-dependent inhibition by ryanodine of caffeine-induced contraction of guinea-pig aortic smooth muscle

We examined how ryanodine interfered with Ca2+-releasing action of caffeine in guinea-pig aorta. Ryanodine (10 microM) decreased the caffeine-induced contraction depending on the time of pretreatment with the agent. The development of ryanodine-effect with time was neither due to a slow access to its binding site nor due to the depletion of stored Ca2+. Ryanodine more potently inhibited the second or the subsequent contraction due to caffeine than the first one even after the agent was removed from the bath after first caffeine. The enhancement of ryanodine-effect depended on the history of Ca2+ release but not on an increase in cytoplasmic Ca2+. The data suggest that an opening of Ca2+ release channel enhances the interaction of ryanodine with the channel.     

Functional role of ryanodine-sensitive Ca2+ stores in acidic pH-induced contraction in Wistar Kyoto rat aorta

Acidic pH induced a contraction in the isolated aorta from Wistar Kyoto rat. The magnitude of contraction was dependent upon the degree of extracellular acidification. The maximum level of contraction observed at pH 6.5 was 84.6 +/- 3.4% of the 64.8 mM KCl-induced contraction. To investigate the role of extracellular as well as intracellular Ca(2+) in acidic pH-induced contraction (APIC), we changed the extracellular pH in the presence of EGTA. Sustained contraction induced by acidic pH in the presence of extracellular Ca(2+) was completely abolished in the presence of EGTA, while a transient but significant contraction was still observed. Ryanodine, a selective ryanodine receptor blocker and cyclopiazonic acid (CPA), an inhibitor of sarco-/endoplasmic reticulum Ca(2+) ATPase, abolished the transient contraction, when pH was decreased in Ca(2+)-free solution. On the other hand, neither xestospongin C, a selective inositol-1,4,5-trisphosphate receptor antagonist nor U-73122, a phospholipase C inhibitor showed this effect. These results suggest the involvement of Ca(2+) release from ryanodine-/CPA-sensitive store of sarcoplasmic reticulum (SR). In normal Ca(2+)-containing solution, ryanodine and CPA did not alter the maximum level of APIC. However, they significantly decreased the rate of rise of APIC. U-73122, suppressed the maximum contraction induced by acidic pH without affecting the rate of rise of APIC, while xestospongin C and U-73343, an inactive analogue of U-73122, had no effect on both parameters of APIC. From these results, it is concluded that acidic pH induces Ca(2+) release from the ryanodine-/CPA-sensitive store of SR and that release provides supportive effect on initiating rapid transient contraction, but not on the sustained contraction, which is entirely due to Ca(2+) influx.     

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Excitation-contraction coupling in isolated adult ventricular myocytes from the rat, dog, and rabbit: effects of various inotropic interventions in the presence of ryanodine

Enzymatically isolated ventricular cells from rats, dogs, and rabbits were electrically stimulated and their membrane potentials were recorded simultaneously with their contractions. Specific pharmacological interventions were used to assess the relative roles of transsarcolemmal Ca2+ entry and the Ca2+ release by the sarcoplasmic reticulum in activating contractions, in these myocytes. We used ryanodine and caffeine to influence Ca2+ release by the sarcoplasmic reticulum, BAY K 8644 and epinephrine to increase Ca2+ entry through Ca2+ channels, and veratridine, ouabain, and monensin to increase Ca2+ entry through Na+-Ca2+ exchange. Ryanodine (1 microM) completely inhibited the shortenings in rat and dog myocytes, but the contractions in rabbit myocytes were much less sensitive to this alkaloid. Similar inhibitory effects of ryanodine were observed in the presence of various inotropic agents with two exceptions: caffeine's effect on the dog myocytes was relatively insensitive to ryanodine and the long-lasting tonic contractions that veratridine triggered in the myocytes of all three species remained completely unaffected by ryanodine. The data indicate that contractile activation in rat and dog ventricular cells is strongly dependent on Ca2+ release from the sarcoplasmic reticulum, while contractility in rabbit myocytes seems to be more dependent on Ca2+ entry through the sarcolemma. The ryanodine-resistant tonic contractions triggered in the myocytes of all three species in the presence of veratridine may be activated by an increased Ca2+ entry via Na+-Ca2+ exchange.     

Defective intracellular Ca(2+) signaling contributes to cardiomyopathy in Type 1 diabetic rats

The goal of the study was to determine whether defects in intracellular Ca(2+) signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca(2+) concentration ([Ca(2+)](i)). The sarcoplasmic reticulum (SR) Ca(2+) store and rates of Ca(2+) release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca(2+) efflux via sarcolemmal Na(+)/Ca(2+) exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca(2+) channel current that triggers Ca(2+) release from the SR. The depression in SR function was associated with decreased SR Ca(2+)-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na(+)/Ca(2+) exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca(2+) signaling caused by alteration of expression and function of the proteins that regulate [Ca(2+)](i) contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na(+)/Ca(2+) exchanger, and unchanged L-type Ca(2+) channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.     

The calcium-ryanodine receptor complex of skeletal and cardiac muscle

[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca2+-induced Ca2+ release from SR vesicles, inhibits [3H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg2+, Ba2+, Cd2+ and La3+ selectively inhibit the skeletal preparation. The toxicological relevance of the [3H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca2+-ryanodine receptor complexes that may play a role in excitation-contraction coupling.     

Some disturbances in the function of ryanodine receptors of spontaneously hypertensive rat cardiomyocytes detected using 4-chloro<Emphasis Type="Italic">m</Emphasis>-cresol

The rate of Ca²⁺ release from the sarcoplasmic reticulum in response to the activation of ryanodine receptors with 4-chlorom-cresol in the cardiomyocytes of three rat strains—spontaneously hypertensive (SHR), normotensive (WKY), and Wistar rats—during 5 weeks of their growth and development was studied aiming to detect the functional differences in the operation of these receptors at various stages of arterial hypertension. In response to 4-chlorom-cresol, a drastic increase in the rates of [Ca²⁺]_i accumulation in SHR myocytes after 17 days of development is recorded versus a decrease in the rates of Ca²⁺ efflux from the sarcoplasmic reticulum of Wistar and WKY rat cardiomyocytes. A correlation between this phenomenon and a genetic defect of ryanodine receptors in SHR rats seems rather unlikely, as the newborn WKY and SHR rats exposed to 4-chlorom-cresol at a concentration range of 0.5–2.0 mM did not display any differences in the rate of sarcoplasmic reticulum Ca²⁺ release. On the other hand, it is possible that the pathological changes in the function of ryanodine receptors manifest themselves later in the ontogenesis. The connection of this phenomenon with an increase in the role of ryanodine receptors in the excitation-contraction coupling in muscle cells and an increase in the calpain expression in SHR rats (absent in the WKY rats) by the age of 3 weeks is discussed. It is assumed that the cleavage of ryanodine receptor subunits by calpain can noticeably intensify the sarcoplasmic reticulum Ca²⁺ release after activation of these receptors without influencing the receptor binding characteristics.     

The effect of ryanodine on isometric tension development in isolated ventricular trabeculae from Pacific mackerel (Scomber japonicus)

An isometric muscle preparation was used to study the inhibitory effect of ryanodine on contractile function in isolated ventricular trabeculae of the Pacific mackerel (Scomber japonicus). Ryanodine (an inhibitor of sarcoplasmic reticulum (SR) function) caused a 20% reduction in peak tension at 20 degrees C, but not 15 degrees C, over the range of frequencies (0.2-3.0 Hz) tested. This indicates that in the absence of a functional SR, the mackerel ventricle can maintain most of its contractile strength utilizing other modes of Ca(2+) delivery to the myofilaments. Ca(2+) flux through the sarcolemmal (SL) L-type Ca(2+)-channels is most likely the predominant pathway for Ca(2+) activation of the myofilaments, although reverse mode Na(+)/Ca(2+) exchange could potentially contribute to a significant extent. High levels of adrenergic stimulation overwhelmed the negative inotropy caused by ryanodine, returning tension to pre-ryanodine levels, further suggesting that the mackerel ventricle can maintain contractile function without Ca(2+) contribution from the SR. These results are discussed within the context of what is known about SR Ca(2+) utilization in rainbow trout and tuna hearts.