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Dietary calcium deficiency is considered to be widespread globally, with published estimates suggesting that approximately half of the world's population has inadequate access to dietary calcium. Calcium is essential for bone health, but inadequate intakes have also been linked to other health outcomes, including pregnancy complications, cancers, and cardiovascular disease. Populations in low‐ and middle‐income countries (LMICs) are at greatest risk of low calcium intakes, although many individuals in high‐income countries (HICs) also do not meet recommendations. Paradoxically, many LMICs with lower calcium intakes show lower rates of osteoporotic fracture as compared with HICs, though data are sparse. Calcium intake recommendations vary across agencies and may need to be customized based on other dietary factors, health‐related behaviors, or the risk of calcium‐related health outcomes. The lack of standard methods to assess the calcium status of an individual or population has challenged efforts to estimate the prevalence of calcium deficiency and the global burden of related adverse health consequences. This paper aims to consolidate available evidence related to the global prevalence of inadequate calcium intakes and associated health outcomes, with the goal of providing a foundation for developing policies and population‐level interventions to safely improve calcium intake and status where necessary. [ABSTRACT FROM AUTHOR]

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation–contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

Vitamin K (VK) is a fat-soluble vitamin that is indispensable for the activation of vitamin K-dependent proteins (VKDPs). It has been shown to play an important role in the proper calcium deposit at the bone level, hindering that on the vascular walls. The deficiency of this vitamin in European populations is frequent and unknown. It is related to several factors, poor dietary intake, altered intestinal absorption or altered production by bacteria, indicating possible dysbiosis. For Vitamin K2 (VK2), there is currently no official reference daily intake (RDI). However, the effects of VK2 on the improvement of health in cardiovascular diseases, on bone metabolism, on chronic kidney diseases have been the subject of research in recent decades. The microbiota in the gastrointestinal tract plays an important role: Bacteroides are primarily capable of synthetizing very long chain forms of menaquinones and, in addition to the bacteria present in the intestinal flora, VK2 is also produced by bacteria used in food fermentation processes. This review provides an update on the current literature regarding the origin of VK2 and its implications in what is called the "calcium paradox", namely the lack of calcium in the bone and its storage in the wall of the vessel.

Calcium supplements are broadly prescribed to treat osteoporosis either as monotherapy or together with vitamin D to enhance calcium absorption. It is still unclear whether calcium supplementation significantly contributes to the reduction of bone fragility and fracture risk. Data suggest that supplementing post-menopausal women with high doses of calcium has a detrimental impact on cardiovascular morbidity and mortality. Chronic kidney disease (CKD) patients are prone to vascular calcification in part due to impaired phosphate excretion. Calcium-based phosphate binders further increase risk of vascular calcification progression. In both bone and vascular tissue, vitamin K-dependent processes play an important role in calcium homeostasis and it is tempting to speculate that vitamin K supplementation might protect from the potentially untoward effects of calcium supplementation. This review provides an update on current literature on calcium supplementation among post-menopausal women and CKD patients and discusses underlying molecular mechanisms of vascular calcification. We propose therapeutic strategies with vitamin K2 treatment to prevent or hold progression of vascular calcification as a consequence of excessive calcium intake.

This study aimed to explore the potential contribution of myofibrils to contractile dysfunction in Ca-paradox hearts. Isolated rat hearts were perfused with Krebs-Henseleit solution (Control), followed by Ca-depletion, and then Ca-repletion after Ca-depletion (Ca-paradox) by Langendorff method. During heart perfusion left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), rate of pressure development (+ dP/dt), and pressure decay (-dP/dt) were registered. Control LVDP (127.4 ± 6.1 mmHg) was reduced during Ca-depletion (9.8 ± 1.3 mmHg) and Ca-paradox (12.9 ± 1.3 mmHg) with similar decline in +dP/dt and -dP/dt. LVEDP was increased in both Ca-depletion and Ca-paradox. Compared to Control, myofibrillar Ca-stimulated ATPase activity was decreased in the Ca-depletion group (12.08 ± 0.57 vs. 8.13 ± 0.19 µmol P/mg protein/h), besides unvarying Mg ATPase activity, while upon Ca-paradox myofibrillar Ca-stimulated ATPase activity was decreased (12.08 ± 0.57 vs. 8.40 ± 0.22 µmol P/mg protein/h), but Mg ATPase activity was increased (3.20 ± 0.25 vs. 7.21 ± 0.36 µmol P/mg protein/h). In force measurements of isolated cardiomyocytes at saturating [Ca], Ca-depleted cells had lower rate constant of force redevelopment ( k , 3.85 ± 0.21) and unchanged active tension, while those in Ca-paradox produced lower active tension (12.12 ± 3.19 kN/m) and k (3.21 ± 23) than cells of Control group (25.07 ± 3.51 and 4.61 ± 22 kN/m, respectively). In biochemical assays, α-myosin heavy chain and cardiac troponin T presented progressive degradation during Ca-depletion and Ca-paradox. Our results suggest that contractile impairment in Ca-paradox partially resides in deranged sarcomeric function and compromised myofibrillar ATPase activity as a result of myofilament protein degradation, such as α-myosin heavy chain and cardiac troponin T. Impaired relaxation seen in Ca-paradoxical hearts is apparently not related to titin, rather explained by the altered myofibrillar ATPase activity. [ABSTRACT FROM AUTHOR]

High calcium intake may increase hip fracture (HF) incidence. This phenomenon, known as the calcium paradox, might be explained by vaccenic acid (18:1 t n-7, VA), the highly specific trans fatty acid (TFA) present in dairy products. First, we ecologically investigated the relationship between 18:1 TFA intake and HF incidence using data from 12 to 13 European countries collected before 2000; then we measured the effects of VA and elaidic acid (18:1 t n-9, EA) on osteoblasts from goldfish scales (tissues very similar to mammalian bone), with alkaline phosphatase as a marker; and finally we measured the effect of VA on mRNA expression in the scales for the major bone proteins type I collagen and osteocalcin. HF incidence was significantly correlated with 18:1 TFA intake in men ( r =0.57) and women ( r =0.65). Incubation with 1 μmol/L VA and EA for 48 h significantly decreased alkaline phosphatase activity by 25% and 21%, respectively. Incubation of scales with 10 μmol/L VA for 48 h significantly decreased mRNA expression for type I collagen and osteocalcin (by about 50%). In conclusion, VA may be causatively related to HF and could explain the calcium paradox. It may be prudent to reduce 18:1 TFA intake, irrespective of trans positions, to prevent HF. [ABSTRACT FROM AUTHOR]

'Calcium paradox' as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles. [ABSTRACT FROM AUTHOR]

BACKGROUND: Vascular calcification and osteoporosis share similar etiopathogenetic mechanisms. Vitamin K2 deficiency could be responsible of the so called "calcium paradox", that is the lack of calcium in the bone and its storage in the vessel wall. These events may have clinically relevant consequences, such as cardiovascular accidents, and bone fractures. AIM: To review the biological function of vitamin K2 metabolism, the main factors related to its deficiency and the consequent clinical significance. DISCUSSION: Vitamin K2 is essential for the function of several proteins, involved in the maintenance of the normal structure of arterial wall, osteoarticular system, teeth, and for the regulation of cell growth. It has been demonstrated to have a pivotal role in the inhibition of vascular foci of calcification, and in the regulation of calcium deposition in the bone. Vitamin K2 deficiency is often subclinic in a large part of healthy population. This deficiency is related to the interaction of various factors, such as the reduced dietary intake, the alteration of intestinal absorption or production, with a possible role of intestinal microbiota and the increased consumption at the vessel wall. CONCLUSIONS: Vitamin K2 deficiency has recently been recognized as a protagonist in the development of vascular calcification and osteoporosis. Data reported so far are promising and, dietary supplementation seems a useful tool to contrast these diseases. However, large studies or solid clinical correlations regarding vitamin K2 deficiency and its pathologic consequences are needed to confirm these preliminary experiences. [ABSTRACT FROM AUTHOR]

Objective: To assess the effect of intermittent custom-made dietary supplementation with Ca, Mg, and vitamin D based on an individual trace element (TE) multielement profile (MP) of the hair and whole blood (WB). Subjects: Initially, we used the hair and WB TE MP to assess Mg, Ca, and Si nutritional status of 48 healthy subjects of both sexes. After the completion of the proposed customized supplementation regimen, 3 perimenopausal women reported for the control TE MP analysis. Before the supplementation, one of them (Subject A) had increased Ca, Mg, and Si in the hair, the other (Subject B) had Ca and Mg increased, and the third one (Subject C) had normal hair Ca and Mg. Methods: TE MP of 41 elements was analyzed in the hair and WB with inductively coupled plasma mass spectrometry (ICP MS). Data on Ca and Mg are also reported for all the healthy subjects. Results: Women have higher Ca and Mg of the hair than men. Counter intuitively, when dietary supplemented with Ca, Mg, Si and vitamin D, hair Ca, Mg and Si decrease in perimenopausal women having initially high Ca, Mg, and Si hair concentrations (calcium paradox). Conclusion: TEMP of the hair is the important indicator of the individual trace element status so that the dietary supplementation can be customized. Indeed, hair is a "memory tissue" due to its unidirectional growth, whereas TE in the blood has to equilibrate with the surrounding biochemical pools. The better bone mineralization may be expected if Ca, Mg, and vitamin D are supplemented simultaneously in accord with an objective individual trace element multielement profile (TE MP) of the hair. [ABSTRACT FROM AUTHOR]

Exposure to low Ca2+ and/or Mg2+ is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca2+ overload and cell death. This phenomenon has been called the ‘Ca2+ paradox’ of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca2+ and Mg2+ is ‘detected’ and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca2+ and Mg2+ that mimic the initial condition of the Ca2+ paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg2+, (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP2), and (v) can be inhibited by Gaq-linked G protein-coupled receptors linked to phospholipase C β-induced hydrolysis of PIP2. Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons ‘detect’ reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia. [ABSTRACT FROM AUTHOR]

Homeostasis in concentration of essential elements and its abnormality due to cancer have been observed for single hair samples by X-ray fluorescence analysis using synchrotron radiation. Ca, Br and K are associated with the cellular signal transduction and were found to have two levels of upper and lower concentrations in hair due to gating their ion channels of hair matrix cells where the channels opened by deficiency of the element. The analysis of patient hair from its root to tip revealed that breast cancer accompanied the characteristic long-period decrease from the upper to the lower level of hair [Ca] that had started 8 to 36 months before finding the cancer; the decreasing profiles were unique to cancer as a competitive process between parathyroid hormone PTH and cancer-induced PTH-related protein by comparison with those for non-cancer subjects. It is concluded that breast cancer originates in disturbance of the signal transduction by the high [Ca] in cells. The observed [Br] in hair gave signs for serious diseases such as renal failure. K deficiency was observed for a half of healthy subjects, similarly to the population of hypertension in Japan. Hair from hypertension patients showed K deficiency, unless taking depressor. Most of elements such as Fe, Cu and Zn in hair were indicators for the liver functions of their storage and excretion. [ABSTRACT FROM AUTHOR]

Although Na+–H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3–-free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3–-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia–reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia–reperfusion-induced myocardial injury. [ABSTRACT FROM AUTHOR]

Abstract: Adaptation to intermittent high altitude (IHA) hypoxia can protect the heart against ischemia-reperfusion injury. In view of the fact that both Ca2+ paradox and ischemia-reperfusion injury are associated with the intracellular Ca2+ overload, we tested the hypothesis that IHA hypoxia may protect hearts against Ca2+ paradox-induced lethal injury if its cardioprotection bases on preventing the development of intracellular Ca2+ overload. Langendorff-perfused hearts from normoxic and IHA hypoxic rats were subjected to Ca2+ paradox (5 min of Ca2+ depletion followed by 30 min of Ca2+ repletion) and the functional, biochemical and pathological changes were investigated. The Ca2+ paradox incapacitated the contractility of the normoxic hearts, whereas the IHA hypoxic hearts significantly preserved contractile activity. Furthermore, the normoxic hearts subjected to Ca2+ paradox exhibited a marked reduction in coronary flow, increase in lactate dehydrogenase release, and severe myocyte damage. In contrast, these changes were significantly prevented in IHA hypoxic hearts. We, then, tested and confirmed our hypothesis that the protective mechanisms are mediated by mitochondria ATP-sensitive potassium channels (mitoKATP) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the protective effect of IHA hypoxia was abolished by 5-hydroxydecanoate, a selective mitoKATP blocker, and significantly attenuated by KN-93, a CaMKII inhibitor. In conclusion, our studies offer for the first time that IHA hypoxia confers cardioprotection against the lethal injury of Ca2+ paradox and give biochemical evidence for the protective mechanism of IHA hypoxia. We propose that researches in this area may lead a preventive regimen against myocardial injury associated with Ca2+ overload. [Copyright &y& Elsevier]

The protective effects of various divalent cations against the irreversible damage of myocardium, a phenomenon termed “the Ca2+-paradox”, were examined in the isolated perfused pigeon heart. All cations examined were added at a concentration of 200 μmol l-1 in the “calcium-free” medium. In hearts perfused with low calcium, upon normal calcium repletion, the maximal recovery of the contractile tension (in the 2nd minute) was approximately 115% and the recovery obtained at the end of reperfusion was 81.5% (compared to the equilibration period value). From the other divalent cations examined, the presence of cobalt, nickel, manganese or barium during calcium depletion powerfully protected the pigeon heart. Upon calcium repletion, the maximal recovery of contractile tension was approximately 60%, 76.5%, 100% and 85%, the recovery estimated at the end of reperfusion was 40%, 12%, 70% and 53%, and the resting tension estimated at the end of reperfusion was 2.69±0.18 g, 6.40±0.50 g, 1.20±0.10 g and 1.90±0.10 g for cobalt, nickel, manganese and barium, respectively. On the contrary, strontium exerted no protective effects. The protective effects were also indicated by reduced total protein and lactate dehydrogenase activity release into the effluent perfusate and maintenance of electrical activity. The effectiveness of the added divalent cations (with the exception of strontium) showed a strong dependence upon their ionic radius. The most potent inhibitors of this phenomenon in the pigeon heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanisms involved in the protective effects of these cations. [ABSTRACT FROM AUTHOR]

To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca2+ requirements for half and maximum activities are 220 µM and 2 mM, respectively. In the absence of Ca2+ the protease is strongly activated by Mn2+ or Sr2+. In the presence of Ca2+, Mn2+ and Sr2+ exhibit a synergistic effect; Mg2+ and Ba2+ have no effect, whereas Co2+, Ni2+ and Cd2+ completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/ calpastatin ratio is lowered during Ca2+ depletion, during Ca2+ repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 µM Mn2+ or Ba2+, in the Ca2+-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca2+ depletion, produces two main peaks of ∼150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2nd min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart. [ABSTRACT FROM AUTHOR]

Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na+]i. The data suggest that taurine efflux is regulated by [Na+]i and cellular osmolality, but not by cellular pH. [ABSTRACT FROM AUTHOR]

The effects of hexarelin, a growth hormone (GH) secretagogue, and human GH on the mechanical and metabolic changes measured in isolated rat hearts submitted to 5 min of Ca deprivation followed by reperfusion with Ca-containing medium, the so-called calcium paradox phenomenon, were studied. Hexarelin (80 μg/kg bid, subcutaneously) administered for 7 d to male rats effectively antagonized the sudden increase in resting tension measured in vitro on Ca repletion. Moreover, during Ca repletion the release of creatine kinase activity (an index of cell damage) in the perfusate of these hearts was reduced up to 40% compared with controls. By contrast, administration of hexarelin for 3 d or GH (400 μg/kg bid, subcutaneously) for 7 d did not affect the mechanical and metabolic alterations induced by the calcium paradox. To assess its direct and acute cardiac effects, hexarelin (8 μg/mL) was perfused in vitro in recirculating conditions for 60 min through the hearts of normal rats. In this case, hexarelin did not stimulate heart contractility and failed to prevent ventricular contracture upon Ca readmission, whereas diltiazem, a Ca channel blocker, effectively antagonized the calcium paradox phenomenon. We conclude that short-term in vivo exposure to hexarelin, but not GH, enables cardiac myocyites to prevent cytoplasmatic electrolytic unbalance and to control intracellular Ca gain, two functions largely impaired during the calcium paradox phenomenon. Moreover, because the effect of hexarelin is not acute but dependent on the length of in vivo treatment, we suggest that it requires modifications of myocardiocyte physiology. [ABSTRACT FROM AUTHOR]

• Blebbistatin improves single cardiomyocyte preparation for electrophysiological studies. • Blebbistatin terminates cardiomyocyte contraction to prevent calcium-paradox damage. • Blebbistatin shows no side-effects on action potentials and field potentials. • Blebbistatin improves I Na and action potential recording using automated patch-clamp. • Phenotypes of Brugada syndrome could be recapitulated using automated patch-clamp. Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation–contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and I Na recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput. [ABSTRACT FROM AUTHOR]

Fingolimod (FTY720) inhibits Ca 2+ -permeable, Mg 2+ -sensitive channels called transient receptor potential melastatin 7 (TRPM7), but its effects on Ca 2+ paradox (CP) - induced myocardial damage has not been evaluated. We studied the effect of FTY720 on CP-induced myocardial damage and used other TRPM7 channel inhibitors nordihydroguaiaretic acid (NDGA) and Mg 2+ to test if any effect of FTY720 was via TRPM7 inhibition. Langendorff-perfused Wistar rat hearts were treated with FTY720 or NDGA and subjected to a CP protocol consisting of Ca 2+ depletion followed by Ca 2+ repletion. Hearts of rats pre-treated with MgSO 4 were also subjected to CP. Hemodynamic parameters were measured using an intraventricular balloon, and myocardial infarct size was quantified using triphenyltetrazolium chloride stain. TRPM7 proteins in ventricular tissue were detected using immunoblot analysis. FTY720, but not NDGA, decreased CP-induced infarct size. Both FTY720 and NDGA minimized the CP-induced elevation of left ventricular end-diastolic pressure, but only FTY720 ultimately improved ventricular developed pressure. Mg 2+ pre-treatment had no effect on CP-induced infarct size, nor hemodynamic parameters during CP, nor the level of TRPM7 protein expression in ventricular tissue. Overall, FTY720 attenuated CP-induced myocardial damage, with potential therapeutic implications on Ca 2+ -mediated cardiotoxicity; however, the cardioprotective mechanism of FTY720 seems to be unrelated to TRPM7 channel modulation.

We analyzed total Ca handling of the left ventricle (LV) in the mildly failing heart preparation induced by a temporary intracoronary Ca overloading intervention in eight excised and cross-circulated canine hearts. This Ca intervention consisted of interruption of coronary blood perfusion by Ca-free oxygenated Tyrode perfusion for 10min followed by high-Ca (16 mmol/l) oxygenated Tyrode perfusion for 5 min. This intervention decreased the LV contractility index, E max (end-systolic maximum elastance), by 40% after restoration of the blood cross-circulation. We expected a Ca overload or paradox failing heart resembling the postischemic stunned heart and being characterized by an increased O2 cost of E max. However, LV O2 consumption under mechanically unloading conditions decreased by 30% from control without increasing the O2 cost of E max. To obtain a mechanistic view of this failing heart, we investigated cardiac total Ca handling by our integrative analysis method. In this method, we obtained the internal Ca recirculation fraction (RF) from the decay beat constant of the postextrasystolic potentiation following each sporadic spontaneous extrasystole in these failing LVs. We combined the RF with the decreased E max and the unchanged O2 cost of E max in our recently developed formula of total Ca handling. We found that these failing LVs had a slightly but significantly increased RF accompanied by either a slightly increased futile Ca cycling or a slightly decreased Ca reactivity of E max, or both. Any of these three possible changes can account for the unchanged O2 cost of E max. This result indicates that the present mildly failing heart has not yet fallen into a typical Ca overload or paradox by the temporary Ca overloading intervention. [ABSTRACT FROM AUTHOR]

The aim of the study was to measure with microbeam PIXE elements such as Na, K, Mg, and Ca in cardiac tissue after various treatments in vivo, which affect the cardiomyocyte integrity. It was assumed that local deviations from normal electrolyte levels indicate the degree of cardiac damage. The first step in this feasibility study was comparison of severely damaged cardiac tissues with controls. Severe cardiac damage was introduced by the so-called Ca paradox. Experiments were performed with isolated rat hearts, perfused retrogradely with an oxygenated crystalloid buffer. Results indicated that severe cardiac damage was accompanied with almost complete disappearance of the normal intracellular electrolyte composition as a result of the loss of membrane integrity. Identifications of smaller and more locally present ischemic damages on basis of altered electrolyte levels appeared to be feasible. However, the prerequisite was that the mobility of electrolytes be kept under control during tissue sampling and sample preparation, when physiological mechanisms stop to maintain gradients. [ABSTRACT FROM AUTHOR]

Objective: Cation non-selective conductances which are induced upon removal of extracellular divalent cations have been identified in cardiac and other cells. We have examined whether the conductance identified in cardiac myocytes mediates an increase in intracellular Na+ (Nai+) and have tested the ability of drugs to prevent this influx. Methods: Rat single ventricular myocytes at 22°C were voltage-clamped in whole-cell mode to measure membrane currents or were loaded with SBFI to measure Nai+. Results: Removal of extracellular Ca2+ (Cao2+) and Mg2+ (Mgo2+), which induced a current with reversal potential of −10 mV, also caused an increase in SBFI fluorescence ratio (340/380 nm). These changes were reversible on repletion of Cao2+ and/or Mgo2+. They could not be prevented by nifedipine, indicating that they were not mediated by L-type Ca2+ channels. Both increases in non-selective conductance and in Nai+ were prevented by trivalent cations (Dyo3+, Gdo3+ or Lao3+; 100 μM) or reduced by the aminoglycoside gentamicin. Conclusion: A cation non-selective conductance, different from L-type Ca2+ channels, contributes to the Nai+ accumulation obtained during perfusion with Ca2+/Mg2+-free media, hence also to the Cai2+ overload and cellular damage upon Cao2+ repletion (the Ca2+ paradox). [ABSTRACT FROM PUBLISHER]

The effect of taurine on the cellular distribution of [Ca2+]i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca2+ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca2+-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca2+]; rapidly fell to a level of 3–6 × 10−8M. Subsequent restoration of medium calcium increased [Ca2+]i to level 4–7 times normal. Large increases in [Ca2+]i were observed in most cells and were associated with the development of contracture and bleb formation. Taurine pretreatment (20mM) caused no significant effect on [Ca2+]i during Ca2+ depletion. However, it inhibited excessive accumulation of [Ca2+]i during the Ca2+ repletion. Moreover, taurine treated cells recovered their Ca2+-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment. [ABSTRACT FROM AUTHOR]

The effects of oxygen-derived radical scavengers (ODRS) on the heart was investigated during the calcium paradox. Perfusion with Ca-free medium caused cell separation at the intercalated discs and changes in the endothelial cells. Upon Ca reintroduction, a massive cell damage occurred. The cytosolic enzyme, creatine phosphokinase (CPK), was released in large amounts ( p< 0.001). The tissue adenosine triphosphate (ATP) was reduced to 3.7 μmol/g dry weight from the control value of 21.6 μmol/g dry weight and tissue Ca content was increased threefold. The treatment with Superoxide dismutase (SOD) and catalase (CAT) increased percentage of normal cells (62.2%) compared to nontreated Ca paradox group (0.2%) and caused negligible leakage of CPK. Tissue ATP was preserved (p<0.03), and Ca content was also reduced in the hearts treated with SOD and CAT (p<0.03). The cell membranes and vascular endothelium were well preserved in the hearts treated with SOD and CAT. Boiled SOD and CAT administered were totally ineffective. It is suggested that oxygen-active species may have a role in the Ca paradox injury. [ABSTRACT FROM AUTHOR]

We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant ( P<0.05) decrease in the number of spheres on the mitochondrial membranes occurs during the calcium paradox (58.0 ±4. I/μm vs. control 80.5±6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5 ±5.0 gmm). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs. [ABSTRACT FROM AUTHOR]

It is demonstrated a fast and significant depression in the sarcolemmal (Na,K)-ATPase activity that occurs as early as 25 sec after the onset of Ca depletion, and participates in the development of Ca-paradox in the rat heart. Pretreatment of the animals with 7-oxo-prostacyclin (PG12) 24-48 h prior to the experiment prevented fairly the Ca-depletion-induced depression in (Na,K)ATPase activity and the accompanying structural and functional damage to the heart and sarcolemma during Ca-depletion as well as the development of Ca-paradox during the subsequent Ca-repletion. Pretreatment with PGI, was chosen intentionally because previous experiments revealed, that in its late effect the drug is acting via stabilizing the membranes due induction of high activity of (Na,K)-ATPase that has increased affinity to ATP. From results obtained the following may be concluded: If during the phase of Ca-deprivation, the capability of heart sarcolemma to maintain sodium extrusion remains preserved, the expected aggravation of Ca-overload injury to Ca-paradox that would develop during Ca-repletion, may be definitely prevented. Sufficiently preserved (Na,K)-ATPase activity, hand in hand with stabilized sarcolemmal structure, may prevent an accumulation of sodium beneath the sarcolemma and consequently also an overexcessive entry of Ca into the myocytes. [ABSTRACT FROM AUTHOR]

To obtain insight into the relation between the release of heart-type fatty acid-binding protein (H-FABP) and of long-chain fatty acids (FA) from injured cardiac tissue, rat hearts were Langendorff perfused according to the following scheme: 30 min normoxia, 60 min ischemia, 30 min reperfusion, 10 min Ca free perfusion and finally 10 min Ca repletion. During this protocol right ventricular ( Q) and interstitial effluent samples ( Q) were collected at regular intervals. During reperfusion a total of 0.8±0.1 nmol H-FABP but no FA were detected in the effluents. However, during Ca readmission, 45±4 nmol H-FABP (80-90% of total tissue content) was released with an initial (first 3 min) simultaneous release of FA (FA/H-FABP ratio 0.90±0.07 mol/mol). Thereafter, FA release continued at 10-15 nmol per min mainly in Q while the rate of H-FABP release decreased. During Ca repletion, tissue FA content raised rapidly from 168±20 to 1918±107 nmol/g dry weight. These findings suggest that after severe cardiac damage initially FA is released bound to H-FABP, whereas further FA release occurs in a non-protein bound manner. [ABSTRACT FROM AUTHOR]

We investigated the effect of Ca overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca-containing medium following either Ca-containing or Ca-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [H]inositol phosphates ([H]IPs) in the tissues prelabeled with [H]inositol. Ca repletion following Ca-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [H]IPs accumulations. Treatment with ouabain following Ca-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [H]IPs were significantly potentiated by prior Ca-free superfusion instead of Ca-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol. [ABSTRACT FROM AUTHOR]

Translocation of lipids inside mammalian cells is considered to be facilitated by a number of low-molecular weight lipid binding proteins. An overview of these proteins is given, with particular reference to the heart. Three distinct phospholipid transfer proteins specifically stimulate the net transfer of individual phospholipid classes between membrane structures. In rat cardiac muscle their content is 15-140 pmol/g ww. Fatty acid-binding proteins (FABP) are abundantly present in tissues actively involved in the uptake or utilization of long-chain fatty acids, such as intestine, liver and heart. The four distinct FABP types now identified show a complex tissue distribution with some tissues containing more than one type. Heart (H-) FABP comprises about 5% of the cytosolic protein mass; its content in rat heart is 100 nmol/g ww. Immunochemical evidence has been obtained for the presence of H-FABP in several other tissues, including red skeletal muscle, mammary gland and kidney. Beside long-chain fatty acids FABP binds with similar affinity also fatty acyl-CoA and acyl-L-carnitines. In heart the latter compound may be the primary ligand, since normoxic acyl-L-carnitine levels are several fold higher than those of fatty acids. In addition, H-FABP was found to modulate cardiac energy production by controlling the transfer of acyl-L-carnitine to the mitochondrial β-oxidative system. H-FABP may also protect the heart against the toxic effects of high intracellular levels of fatty acid intermediates that arise during ischemia. [ABSTRACT FROM AUTHOR]

Reperfusion of hearts with a Ca-containing medium after a perfusion period in Ca-free medium results in irreversible cell damage (calcium paradox). In this investigation we have studied coronary flow and cyclic AMP and cyclic GMP levels after several periods of Ca-free perfusion in isolated rat hearts. We also investigated the effects of papaverine (Pap), noradrenaline (NA), acetylcholine (ACh) and absence of inorganic phosphate during Ca-free perfusion on coronary flow (CF) and cyclic nucleotide levels. Inability of the heart to recover contractile activity with development of contracture during the reperfusion period was accepted as indicative of the calcium paradox. Ca-free perfusion alone and NA and absence of inorganic phosphate during the Ca-free perfusion period increased CF, whereas Pap and ACh decreased it. However, only Ca-free perfusion and NA elevated cyclic AMP. On the other hand, Pap and ACh increased cyclic GMP (with a transient rise of cyclic AMP in Pap infusion), and absence of inorganic phosphate decreased both cyclic AMP and cyclic GMP. Pap, ACh and absence of phosphate prevented the calcium paradox. Our study suggests that increased cyclic AMP during the Ca-free perfusion may contribute, with the other factors, to the occurrence of the calcium paradox. [ABSTRACT FROM AUTHOR]

Reduction of the Na concentration in the Ca-free perfusion solution reduces the amount of myoglobin released by the cells when Ca is readmitted if sucrose is used to replace NaCl under mild hypothermia. When salts like cholinechloride or LiCl are used instead of sucrose, no protection is seen at any temperature. The temperature threshold above which myoglobin loss sharply increases is lowered by prolonged Ca depletion or by the addition of EGTA to the Ca-free solution. Protection by sucrose does not occur in the presence of EGTA. An increase of cell Na induced by strophanthidin during the Ca depletion phase has no effect on myoglobin release. The exponential decline in twitch tension in the early phase of Ca deprivation has the same half-live ( T) for Ca-free solutions containing 145 mM Na or 35 mM Na (110 mM Li or choline), but its T is prolonged if sucrose is used to replace NaCl. When 5 mM EGTA is added to the Ca-free solutions, the T is shortened and is not changed by the replacement of NaCl with sucrose. The rate of washout of Ca within the first 20 s of Ca depletion has a similar time course in a normal Na or in a Li and low Na solution. In a sucrose and low Na solution the rate of the Ca efflux is reduced. The addition of EGTA increases this rate and abolishes the slowing effect of a sucrose and low Na solution. Therefore myoglobin release during the Ca paradox does not depend on the Na gradient across the sarcolemma. Na, like other cations, probably enhances the displacement of Ca from critical binding sites during Ca-free perfusion, which predisposes the cells to the paradox. [ABSTRACT FROM AUTHOR]

Reperfusion of isolated rat hearts with Ca-containing medium, after a short Ca-free perfusion period, results in irreversible cell damage (calcium paradox). In this investigation the effect of hypothermia during (a) the Ca-free perfusion period and (b) the phase of reperfusion with Ca-containing medium was studied. Failure of the heart to recover mechanical activity, and creatine kinase release were used to define cell damage. Ca-free perfusion was performed at 37°, 30°, 25° and 20°C. Hypothermia during the Ca-free period was unable to prevent the calcium paradox. At 37° C a Ca-free perfusion period of 4 min was sufficient to lead to failure of the hearts to recover mechanical activity and to induce massive enzyme release upon reintroduction of Ca. At 20°C the Ca-free perfusion had to be continued for 25 min to induce failure to recover mechanical activity, and for 80 min to induce massive enzyme release upon reperfusion with Ca-containing medium. Hypothermia (10°-15° C) during the reperfusion phase resulted in a moderate release of creatine kinase. Massive enzyme release occurred as soon as the temperature of the perfusate was raised above 25°C. [ABSTRACT FROM AUTHOR]

Primary cultures of rat myocytes were deprived of oxygen (approximately 5 mm Hg, pO) for 2 h in the presence or absence of calcium and were subsequently incubated for different time periods under normoxic (approximately 120 mm Hg, pO) conditions. Myocyte calcium content was determined at the end of the oxygen-free period and after repletion of oxygen. Lactate dehydrogenase (LDH) release from the cells into the medium was used as an index of cell injury. Cultures deprived of oxygen in the absence of calcium showed a significant decrease in myocyte calcium content at the end of the oxygen-free period. However, the calcium content of myocyte cultures deprived of oxygen in the presence of calcium did not change significantly. The enzyme release of both groups of oxygendeprived cultures was similar to that of controls. A progressive increase in myocyte calcium content was observed 5, 10, 15, and 30 min after repletion of oxygen in both groups. Reoxygenation of cultures subjected to hypoxia in the presence of calcium caused minimal LDH leakage. However, major enzyme release was observed 30 min after oxygen repletion in cultures previously subjected to hypoxia in a calcium-free environment. The present study shows that reoxygenation injury is more severe in myocyte cultures previously deprived of both oxygen and calcium than in cultures deprived of oxygen in the presence of calcium. [ABSTRACT FROM AUTHOR]

The occurrence of arrhythmias was studied in the 'calcium-paradox' model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period. The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60-90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

The occurrence of arrhythmias was studied in the 'calcium-paradox' model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period. The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60-90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

The aims of the present study were to establish if micromolar manganese (50 μmol) may substitute for calcium during calcium-free perfusion and also to assess if manganese substitution for calcium may ensure tissue protection by verapamil (1.0 mg/l). Two injury levels (minimal and total calcium paradox) caused by different volumes (5 ml and 45 ml) of calcium-free perfusion (5 min) prior to calcium repletion (15 min) were examined in a normothermic isolated rat heart model. The presence of manganese during calcium-free only (5 min) or the presence of manganese prior to (5 min), during (5 min), and following (5 min) calcium-free perfusion conferred considerable protection as assessed by enzymatic, physiological and metabolic parameters in both the total and minimal calcium paradox models. The presence of verapamil prior to (5 min), during (5 min) and following (5 min) calcium-free perfusion combined with the presence of manganese conferred a further protection, particularly in the total paradox. It is concluded from the study that 50 μmol of manganese, although effective in tissue protection, cannot substitute totally for the loss of calcium during calcium-free perfusion and that manganese may substitute for the micromolar calcium needed for tissue protection by verapamil. [ABSTRACT FROM AUTHOR]

In a graded model of the calcium paradox phenomenon (minimal and total) the presence of the β-blocker propranolol (5·10M) in the perfusion media (10 min prior to, during and 5 min following calcium-free perfusion) has no effect upon tissue injury. Propranolol pretreatment (three days prior to the experiments) significantly reduced the myocardial enzyme release during calcium repletion in the minimal calcium paradox. The presence of the α prazosin (1·10M) in the perfusion media (10 min prior to, during and 5 min following calcium-free perfusion) afforded no protective effects. It is concluded that the release of endogenous catecholamines may not be an important factor contributing to myocardial injury in the calcium paradox, and that consequently β- or α-adrenergic blockade has little if any protective properties in this form of myocardial injury. [ABSTRACT FROM AUTHOR]

Reperfusion of an isolated heart with a calcium-containing solution after a short calcium-free perfusion may result in irreversible cell damage: the calcium paradox. In this investigation the effect of hypothermia during reperfusion with calcium-containing solution on the calcium paradox damage in the isolated rat heart was studied. In addition, the effect of pre-cooling the heart during the calcium-free period was investigated. Creatine kinase release was used to define cell damage. Normothermic (37°C) calcium-free perfusion followed by normothermic reperfusion with calcium-containing solution resulted in a massive release of CK. When the normothermic calcium-free perfusion was followed by hypothermic (10°C) calcium-containing reperfusion, CK release was reduced by 20% (P<0.005). This CK release during reperfusion was further reduced by 55% and 80% when the normothermic calcium-free perfusion was followed by 5 or 10 min respectively of hypothermic calcium-free perfusion prior to the hypothermic calcium-containing reperfusion. The results show that hypothermia during the period of calcium repletion retards the sequence of events which ultimately results in release of large amounts of intracellular components. [ABSTRACT FROM AUTHOR]

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP. [ABSTRACT FROM AUTHOR]

We observed changes in the performance of isolated right ventricle strips taken from rats when calcium was repleted following various periods of calcium depletion in order to study certain phenomena, such as the calcium paradox, in this preparation. Furthermore, to assess the possible role of calmodulin in this myocardial damage, the effects of known calmodulin inhibitors such as trifluoperazine and chlorpromazine on the contractility and resting tension were studied by means of the calcium repletion after a calcium-depleted period of 12 min. The temperature was kept at 37°C, and the muscle strips were stimulated electrically at a rate of 0.25 Hz. When there was a calcium-depleted period of longer than 8 min, a marked increase in resting tension was observed and reached maximum at 2 to 4 min. The recovery of peak developed tension and peak positive or negative dT/dt worsened as the duration of the calcium depletion was longer. These findings indicate the massive intracellular calcium influx by the calcium reintroduction and the myocardial damage induced by the calcium overload as observed in isolated whole hearts. Treatment with trifluoperazine (1-5 μM) and chlorpromazine (1-5 μM) did not inhibit a rise in resting tension significantly after the calcium repletion, except for 5 μM of both drugs at 6 min. Trifluoperazine significantly improved the recovery of the contractility (developed tension and dT/dt), whereas the protective effect of chlorpromazine was not obtained. These results suggest that the depression of calmodulin activity is beneficial in the prevention of myocardial damage produced by calcium repletion, although there is a difference in the effect of the calmodulin inhibitors, trifluoperazine and chlorpromazine. [ABSTRACT FROM AUTHOR]

The effects of the presence or absence of calcium in the cardioplegic perfusate were studied utilizing the isolated blood perfused dog heart preparation. Hearts were subjected to two hours of arrest at 27°C followed by 90 minutes of normothermic reperfusion. Perfusates with or without 2.52 mM calcium chloride were delivered at 15 minute intervals during arrest at a perfusion pressure of 100 mm Hg. Both calcium and calcium free perfusates resulted in decreases in tissue calcium concentration measured at the end of arrest. Tissue magnesium concentrations did not change with either perfusate. Coronary vascular resistance was increased with early perfusions in the calcium perfused group relative to the calcium free group. Systolic and diastolic performance, high energy phosphate values, coronary blood flow and myocardial oxygen consumption were altered by the arrest-reperfusion sequence but no differences between calcium and calcium free groups were seen. Thus, no evidence for deleterious effects of calcium free perfusion was present after two hours of global cardiac arrest followed by reperfusion. The data indicate the absence of the calcium paradox under conditions simulating clinical cardioplegia and supports clinical data indicating that the addition of calcium to the cardioplegic solution is unnecessary. [ABSTRACT FROM AUTHOR]

The effects of omission of Mg during Ca-free perfusion (3 min) of either spontaneously beating and electrically stimulated rat hearts were studied. Ca-free perfusion per se induced cardiac arrest and coronary vasodilation, and increased intrinsic pulse rate of the heart. Upon reperfusion with Ca, cardiac function was lost and parallelled by a sudden and massive release of cellular constituents ('calcium paradox'). Mg-free perfusion evoked effects opposite from Ca-free, with exception of heart rate which was increased. During Ca−Mg-free perfusion the electrocardiogram became irregular within 30 s, and this effect was followed by transient 'spasmodic contractions'. The effects of normal reperfusion were indistinguishable from those observed after Ca-free perfusion in the presence of Mg. Addition of Mn or La to the Ca−Mg-free perfusion medium completely inhibited the induction of electrical irregularities and spasmodic contractions. The typical effects of Ca−Mg-free perfusion are discussed in terms of Mg−Ca and Mg−K interactions at the sarcolemmal surface. It was concluded that our results may contribute indirectly to an explanation of the protective effect of high Mg-concentrations during Ca-free as well as ischemic perfusion of mammalian hearts. [ABSTRACT FROM AUTHOR]

ABSTRACT- Our study was designed to determine the calcium and oxygen paradoxes in isolated rat hepatocytes, and to evaluate the cytoprotective effects of taurine on hepatocytes during oxygenation, hypoxia, and on these paradoxes. The calcium and oxygen paradoxes were clearly revealed in isolated rat hepatocytes. As the formation of the superoxide radical was accelerated by the hyperbaric conditions and the calcium ions, drugs such as superoxide dismutase and a calcium channel blocking agent (verapamil), prevented these paradoxes. Taurine decreased the oxygenation-induced lipid peroxidation of hepatocytes, and prevented the hypoxia-induced hepatocyte death in a calcium-containing medium, but not in a calcium-free medium. Taurine also protected the hepatocytes from injuries associated with the calcium and oxygen paradoxes due to the inhibition of sudden calcium influx into the hepatocytes. [ABSTRACT FROM AUTHOR]

The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism. [ABSTRACT FROM AUTHOR]

The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37°C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22°C and 27°C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37°C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24±2.49 IU·g dry wt, and the total amount of protein lost was 70.37±0.73 mg·g dry wt, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37°C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts. [ABSTRACT FROM AUTHOR]

The effects of calcium (Ca) on a hyperkalemic cardioplegic solution for continuous cardioplegia were examined in an isolated perfused working rat heart model. The coronary arteries were perfused with a modified Krebs-Henseleit bicarbonate buffer (K-H) solution, containing various concentrations of Ca(0.1, 0.6, 1.2, and 2.5 mmol/l) and a high concentration of potassium (20 mmol/l), for 180 min, after which cardiac arrest was induced at 37°C for 180 min. Cardiac function and creatine kinase (CK) were measured. In the control group, K-H solution was infused in place of the cardioplegic solution, and cardiac arrest was not induced. No significant differences were observed between the groups infused with the K-H solution containing Ca concentrations of 0.6, 1.2, and 2.5 mmol/l in the percent recovery of aortic flow (82.1±2.9%, 80.6±2.0%, and 71.5±3.7% (mean±SEM) respectively) or in the recovery of other indices of cardiac function, or in CK leakage. There were also no significant differences in the recovery of cardiac function and CK leakage between these groups and the control group. In the Ca 0.1 mmol/l group, however, the characteristic Ca paradox was observed. These findings suggest that if the Ca concentration in a cardioplegic solution is higher than 0.6 mmol/l during continuous cardioplegia, excellent cardioprotective effects will be achieved. [ABSTRACT FROM AUTHOR]

Objective: Although it is widely accepted that the hypercontraction provoked by the influx of Ca2+ on return to normal Tyrode solution after a period of Ca2+ depletion (the calcium paradox) contributes to the damage to the cell membrane, it remains possible that the Ca2+ overload activates intracellular enzymes that are important in the initial degradation of the cell membrane. This possibility was examined in this work. Experimental design: The effect of Ro 31-4493, a phospholipase A2 inhibitor, upon protein loss, hypercontracture, and ultrastructural changes in Langendorff perfused guinea pig hearts during the calcium paradox was studied. Because the degree of Ca2+ overload might also be affected, the action of the drug on the resting and action potentials and the whole cell currents through the L-type Ca2 channels and the changes in [Ca2+]i in isolated guinea pig ventricular myocytes were also studied. Results: Ro 31-4493, at 10-50 μM, inhibited the release of proteins from guinea pig Langendorff perfused hearts during the calcium paradox in a dose dependent way. This protective effect required preincubation with the agent before the Ca2+ depletion. No significant effect upon the contractile behaviour, as recognised from the sustained increase in intraventricular pressure on Ca2+ repletion, was produced. However, structural changes suggest that the extent of hypercontraction, the disruption of cell membrane and the release of mitochondria was less in treated hearts. Ro 31-4493 produced no change in the resting and action potentials or the behaviour of the L-type Ca2+ channels with either Ca2+ or Na+ as the charge carrier, while measurements of [Ca2+]i indicated a similar Ca2+ overload in both treated and untreated hearts. Conclusion: The effects of Ro 31-4493 are inconsistent with mechanical consequences of hypercontraction upon the cell membranes, being solely responsible for the protein loss during the calcium paradox and suggest that activation of a Ca2+ dependent phospholipases may be an important step.Cardiovascular Research 1994;28:650-655 [ABSTRACT FROM PUBLISHER]

Objective: The aim was to investigate the effects of raising intracellular taurine on the intracellular sodium activity (aNai) in isolated guinea pig ventricular myocytes, and the effect of procedures that raise intracellular sodium on taurine concentration in the perfused guinea pig ventricular tissue. Methods: Taurine was introduced into the sarcoplasm of isolated ventricular myocytes, either during cell isolation or by diffusion from a penetrating micropipette, and the effect on aNai was measured using an ion sensitive microelectrode. Guinea pig hearts, mounted on a Langendorff apparatus, were perfused with a variety of physiological media and the level of taurine in the ventricles determined using high pressure liquid chromatography. Results: An increase in intracellular taurine caused by its presence during cell isolation or by diffusion from a micropipette significantly reduced the aNai of isolated myocytes at rest, during perfusion with Ca depleted solutions, or on inhibition of the Na pump. In the guinea pig ventricles, taurine at 13.0(SEM 0.6) mmol·kg−1 wet weight comprised up to 45% of the free amino acids; since plasma taurine was 64(13) μmol·litre−1, this means that in vivo a large outwardly directed gradient for taurine exists (equivalent to a free energy of 13.7 KJ·mol−1). Upon perfusion with Ca,Mg free Tyrode solution (which raises intracellular sodium markedly), a time dependent loss of taurine occurred. Both the rate of loss and the total amount lost were increased when the Na pump was also inhibited. This loss of tissue taurine was not due to release from dead or lysed cells, as it was antagonised by procedures known to reduce the rise of aNai during Ca depletion, was inhibited by β alanine (an inhibitor of taurine transport), and the fall in tissue taurine was not correlated with the appearance of lactate dehydrogenase in the effluent. Conclusions: The data from isolated myocytes and perfused guinea pig hearts were consistent with the presence of a Na/taurine symport which is activated to cause efflux of Na and taurine when either rise above their physiological level.Cardiovascular Research 1992;26:897–905 [ABSTRACT FROM PUBLISHER]