Search

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]

Cardiovascular disease (CD) and osteoporosis are frequent diseases in adult population, whose frequency increases with age. Thus, with the increase of life expectancy, they become relevant health care issues. Both ECV and osteoporosis depends on the coexistence of multiple risk factors, and prevention policy stresses in controlling those modifiable. ECV and osteoporosis have common etiological factors involving cholesterol biosynthesis and lipid oxidation, whose elucidation supports a relevant research topic. Skeletal growth and maintenance depends on the balance between bone formation and resorption achieved through the process of bone modelling and bone remodelling, controlled by bone tissue cells (osteoblasts --OB-, osteoclasts --OC- and osteocytes), the OB are responsible for bone formation, OC of bone resorption, while osteocytes are key pieces of the bone mechanic sensorial network. The main links between CVD and osteoporosis are the key role of the mevalonate pathway in cholesterol synthesis and OC activation, and the role of oxidised lipids for atherosclerosis development and the conversion of OB-precursor cells in adipocites, reducing OB formation and favouring osteoporosis. In the light of these issues, cholesterol-lowering and antioxidant substances can exert bone protector effects, and the search of drugs effective on both targets support a current research direction, outstanding in the prevention or treatment of such diseases. [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]