Effects on infarct size of reperfusion and pretreatment with β-blockade and calcium antagonists

Summary The mass of tissue at risk and the myocardial infarct developed was studied in dogs subjected to either 24-h occlusion of the left anterior descending coronary artery or 2-h occlusion followed by 22-h reperfusion. The mass of tissue at risk was defined under anaesthesia at the time of occlusion using the microsphere technique. Twenty-four hours later the hearts were removed, sliced transversely and stained with 2,3,5-triphenyltetrazolium chloride to define the infarcted tissue. All myocardial tissue was mapped and cut into small pieces for weighing and radioactive counting. Radioactivity was present in all tissue, including the infarct. In the centre of the the infarct, counts remained low and then increased very rapidly with distance just beyond the edge. Tissue at risk from infarction was taken as that with less than 15% of the peak left ventricular (non-ischaemic) counts.A linear relationship was found between the mass of the left ventricular infarct and the left ventricular mass of tissue at risk. The effect of 22 hours reperfusion was examined by this method and expressed by a regression equation. There was a significant decrease in slope for the regression line of the reperfusion data, (p<0.05, analysis of covariance), indicating less infarcted tissue for each gram of underperfused tissue.None of the drug pretreatments explored had any effect on infarct size in the 24-h occlusion model. With reperfusion, propranolol and flunarizine diminished infarct size compared with reperfusion only (p <0.05 for reduced slope, the new slope being not significantly different from zero). The effect of diltiazem was not so marked. Thus infarct size can be reduced with pretreatment, as long as the myocardium is reperfused.S. Torr and M. Main were supported by grants from the Science and Engineering Research Council, UK; A.J. Drake-Holland was supported by a fellowship from the Janssen Research Foundation.M.I.M. Noble is supported by the Garfield Weston Trust as the Weston Professor of Cardiovascular Medicine.     

Does calcium aggravate and cause hypertension?

Albert Szent-Gyorgyi around 30 years ago proposed that "ions were the powerful tools of life as it developed in oceans". The purpose of this fundamental concept was to divert the attention of investigators towards the basic role of cations such as Na, K, Ca and Mg in muscle contraction. There is now sufficient evidence to support this prediction. Until recently the strongest evidence pointed to a positive relationship between raised Ca and blood pressure. More recent evidence claimed that increased dietary Ca can lower blood pressure and populations taking lowered dietary Ca, have a higher incidence of hypertension. It has been suggested that in susceptible persons, in the presence of high Na intake, Ca accumulation in the arterial cell becomes rapid due to impaired Na-Ca exchange causing an early rise in blood pressure. This is possibly due to abnormal handling of Ca by the smooth muscle cells in most forms of hypertension. Clinical experimental and epidemiologic studies showed that Ca not only mediates arterial smooth muscle contraction but excess of Ca can also cause an increase in peripheral vascular resistance leading to essential hypertension. Ca entry blockers such as verapamil and nifedipine which reduce the influx of Ca into the arterial smooth muscle cell have been successfully used in the management of these patients. The effect of Ca on blood pressure could be independent of other factors. Recent studies strongly favour a significant relationship between raised serum Ca and high blood pressure. Such a relationship has also been described between dietary Ca, 24 urinary Ca and high blood pressure. A large number of studies, particularly from the USA, contradicting the above view, suggest that the rate of Ca flux rather than the absolute quantity that enters the cell, is the deciding factor in arterial smooth muscle contraction. These investigators in the last 6 years have studied a large area of north America and suggested that Ca deficiency rather than an excess is the cause of hypertension. Persons consuming less than 300 mg Ca per day have 11-14% risk of developing hypertension compared to person taking 1200 mg per day of dietary Ca and exposed to only 3-6% risk.(ABSTRACT TRUNCATED AT 400 WORDS)     

Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Bones and Crohn＇s：No benefit of adding sodium fluoride or ibandronate to calcium and vitamin D

AIM: To compare the effect of calcium and cholecalciferol alone and along with additional sodium fluoride or ibandronate on bone mineral density (BMD) and fractures in patients with Crohn’s disease (CD).METHODS: Patients (n =148) with reduced BMD (T-score < -1) were randomized to receive cholecalciferol (1000 IU) and calcium citrate (800 mg) daily alone(group A, n = 32) or along with additional sodium fluoride (25 mg bid) (group B, n = 62) or additional ibandronate (1 mg iv/3-monthly) (group C, n = 54). Dual energy X-ray absorptiometry of the lumbar spine (L1-L4) and proximal right femur and X-rays of the spine were performed at baseline and after 1.0, 2.25 and 3.5 years. Fracture-assessment included visual reading of X-rays and quantitative morphometry of vertebral bodies (T4-L4).RESULTS: One hundred and twenty three (83.1%) patients completed the first year for intention-to-treat (ITT) analysis. Ninety two (62.2%) patients completed the second year and 71 (47.8%) the third year available for per-protocol (PP) analysis. With a significant increase in T-score of the lumbar spine by +0.28 ± 0.35 [95% confidence interval (CI): 0.162-0.460, P < 0.01], +0.33 ± 0.49 (95% CI: 0.109-0.558, P < 0.01), +0.43 ± 0.47 (95% CI: 0.147-0.708, P < 0.01) in group A, +0.22 ± 0.33 (95% CI: 0.125-0.321, P < 0.01); +0.47 ± 0.60 (95% CI: 0.262-0.676, P < 0.01), +0.51 ± 0.44 (95% CI: 0.338-0.682, P < 0.01) in group B and +0.22 ± 0.38 (95% CI: 0.111-0.329, P < 0.01), +0.36 ± 0.53 (95% CI: 0.147-0.578, P < 0.01), +0.41 ± 0.48 (95% CI: 0.238-0.576, P < 0.01) in group C, respectively, during the 1.0, 2.25 and 3.5 year periods (PP analysis), no treatment regimen was superior in any in- or between-group analyses. In the ITT analysis, similar results in all in- and between-group analyses with a significant in-group but non-significant between-group increase in T-score of the lumbar spine by 0.38 ± 0.46 (group A, P < 0.01), 0.37 ± 0.50 (group B, P < 0.01) and 0.35 ± 0.49 (group C, P < 0.01) was observed. Follow-up in ITT analysis was still 2.65 years. One vertebral fracture in the sodium fluoride group was detected. Study medication was safe and well tolerated.CONCLUSION: Additional sodium fluoride or ibandronate had no benefit over calcium and cholecalciferol alone in managing reduced BMD in CD.     

Does the response of bone mass to calcium supplements depend on calcium absorption efficiency?

OBJECTIVE: Calcium supplements can reduce bone resorption and slow bone loss after the menopause, but these effects may be limited by poor intestinal absorption. Since the increase in blood ionised calcium and decrease in serum parathyroid hormone after a calcium load are diminished in patients with poor calcium absorption, we aimed to see whether the response of bone mineral content (BMC) to calcium is related to initial calcium absorption. DESIGN: We retrospectively examined the changes in forearm BMC in 164 patients (139 women and 25 men) receiving calcium therapy alone for low bone density in a university hospital. METHODS: BMC was measured in a Molsgaard single energy absorptiometer and calcium absorption in a single blood sample 1 h after a dose of 5 microCi (45)Ca in 20 mg calcium carrier. Results were analysed by simple and multiple regression analysis. RESULTS: Mean forearm BMC did not change significantly over the mean 43 (S.D., 33) months of treatment (1.023 (0.247) to 1.017 (0.246) g/cm). The annual percentage of change was positively related to both body weight (r=0.180; P=0.020) and radiocalcium absorption (r=0.185; P=0.017). Multiple linear regression confirmed that both variables contributed to the change in BMC (P=0.023 and 0.019 respectively). The mean annual percentage of change in BMC on calcium therapy was not related to age, initial BMC, serum 1,25-dihydroxyvitamin D or fasting urinary calcium/creatinine ratio. CONCLUSIONS: These results support our earlier studies which suggest that poor calcium absorption limits the response of bone to calcium supplements.     

Who should receive calcium and vitamin D supplementation?

Combined calcium and vitamin D supplementation is recommended in the prevention and treatment of osteoporosis. Until recently, supplementation was perceived as harmless without adverse effects. However, recent meta-analyses have provided evidence suggesting that calcium supplements, whether or not in combination with vitamin D, may be associated with cardiovascular risks. Although this finding constitutes a safety signal that has to be taken seriously, these data have to be interpreted with some caution. Current data do not allow definite conclusions to be drawn, but require further independent confirmation, since in numerous large studies, combined calcium and vitamin D supplementation did not increase cardiovascular events, even in the most frail and elderly populations. Nevertheless, it seems appropriate to correct calcium deficiency preferably by enhancing dietary intake and to target supplementation on individuals at high risk of fracture or in whom calcium and vitamin D deficiency is highly prevalent. Other trials have shown an increased risk of falls and fractures with annual oral administration of high dose of vitamin D. Therefore, supplementation with more frequent, lower doses is preferred. Yet, the optimal dosing schedule is unknown and needs further study. In order to correct age-associated secondary hyperparathyroidism and to prevent osteoporotic fractures, a daily dose of 1,000-1,200?mg calcium and 800?IU vitamin D is recommended in elderly or institutionalised people, patients with established osteoporosis and individuals on glucocorticoids.     

What is behind the calcium? The relationship between calcium and necrotic core on virtual histology analyses

We read with great interest the investigative paper by Misselet al.¹ We would like to thank the authors for furthering ourknowledge in this field with their continued production of high-quality     

Changes in the pharmacologic treatment of hypertension in the department of veterans affairs 1997–1999: decreased use of calcium antagonists and increased use of β-blockers and thiazide diuretics

Older studies of antihypertensive treatment have shown that prescribing patterns are not consistent with recommendations from expert national panels. We determined whether prescribing patterns for antihypertensive drugs changed recently in the largest integrated health care system in the United States. Specifically, we determine 1) patterns of antihypertensive medication use at all Department of Veterans Affairs (VA) medical facilities for fiscal years 1997 to 1999, 2) the cost of this care, and 3) savings associated with changes in treatment patterns. Data were aggregated by individual medication as well as by antihypertensive drug class. Estimates of VA national antihypertensive drug costs are based on the median cost and the number of units for each dosage form of each medication dispensed at all facilities.At VA medical facilities, calcium antagonist use went from 33% to 29.3% of antihypertensive treatment days between 1997 and 1999, angiotensin converting enzyme (ACE) inhibitor/angiotensin receptor blocker (ARB) use from 36.4% to 36.8%, β-blockers from 19.1% to 21.1%, and thiazide diuretic use from at 11.5% to 12.8%. If treatment patterns had remained the same between 1997 and 1999 in terms of the proportion of medications from each drug class, an additional six million dollars would have been spent on antihypertensive medications in 1999.Although calcium antagonists and ACE inhibitors/ARB remained the most commonly dispensed antihypertensives at VA facilities from 1997 to 1999, there was a proportional decrease in calcium antagonist use and an increase in the use of thiazide diuretics and β-blockers. These changes were consistent with improved compliance with VA national guidelines. The cost implications of these changes in practice patterns were considerable.     

The influence of endothelium on the action of PGF2α, and some dihydropyridine-type calcium antagonists in porcine basilar arteries

Summary Vascular endothelium modulates the effect of various vasoconstricting mediators as well as the affinity of dihydropyridine-type calcium entry blockers. To further investigate this influence, vasoconstriction by PGF₂ as opposed to KCl and the affinity of nitrendipine and some related 3-ester side-chain derivatives were determined in isolated porcine basilar arteries in the presence and in the absence of intact endothelium, as well as in the presence of methylene blue. Treatment with methylene blue or mechanical endothelial damage increased the contractile work of basilar arteries stimulated by PGF₂ and reduced the affinity of the dihydropyridines in such precontracted vessels. Both experimental conditions resulted in nearly the same effect. In addition, the degree of intact endothelium, as determined by substance-P-induced vasodilation, significantly correlated with the corresponding efficacy of all dihydropyridines examined. In contrast, KCl-mediated contractions remained unchanged. It is suggested that the endothelium (probably due to the production and release of endothelium-derived vasorelaxing factors, such as EDRF and/or prostacyclin) may attenuate PGF₂ induced transmembrane calcium influx through receptor operated calcium channels, whereas potential operated calcium channels seems to be unaffected.Dedicated to Prof. Dr. rer. nat. Dr. med. Ernst Mutschler on the occasion of his 60th birthday     

Total and ionized calcium and magnesium are significantly lowered in drug-naïve depressed patients: effects of antidepressants and associations with immune activation

Metabolic Brain Disease - Major depressive disorder (MDD) is associated with alterations in calcium (Ca) and magnesium (Mg), as well as circulating pro- and anti-inflammatory cytokines....     

Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in Β TC3 cells and rat pancreatic beta cells

Summary The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open probability of single ATP-sensitive K⁺-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (<5 min). The influence of repaglinide and glibenclamide on the ATP-sensitive K⁺ current was studied using the whole-cell patch clamp configuration. A half-maximal steady-state inhibition of the ATP-sensitive K⁺ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca²⁺ imaging on single TC3 cells we found that repaglinide and glibenclamide induced a concentration-dependent increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca²⁺]_i results from Ca²⁺ entry through voltage-dependent L-type Ca²⁺-channels since it is inhibited by verapamil (10 mol/l). The effect of repaglinide and glibenclamide is partly reversible (80%).Abbreviations K⁺ _ATP-channel ATP-sensitive potassium channel - [Ca²⁺]_i intracellular free Ca²⁺ concentration - EGTA ethylene glycol-O,O-bis(2-aminoethyl]-N,N,N,N-tetraacetic acid - IC₅₀ apparent inhibitor constant - repaglinide ((S)-(+)-2-ethoxy-4-[2-((3-methyl-1-[2-(1-piperidinyl) phenyl]-butyl)amino)-2-oxoethyl) benzoic acid - NIDDM non-insulin-dependent diabetes mellitus - TC3 cells transgenic mouse insulinoma tumour cell line     

Serum calcium and cardiovascular risk factors and diseases: the Troms? study.

Total serum calcium levels were measured in 12 865 men and 14 293 women, between the ages of 25 and 97 years, in the Troms? Study during 1994 and 1995. With the use of a sex-specific multiple linear regression model with age, calcium, body mass index, cholesterol, HDL cholesterol, triglycerides, systolic and diastolic blood pressure, and pulse as possible covariates, serum calcium was significantly (P<0.001) and positively associated with systolic and diastolic blood pressure, serum cholesterol, and HDL cholesterol in both sexes. A similar but weaker association was observed between serum calcium and triglycerides in men (P<0.01). In all age groups, serum calcium levels were higher in men with a history of myocardial infarction than in those without, and the difference was significant (P<0.0001) in a linear regression analysis adjusted for age. When all the other variables were also included in a logistic regression model, serum calcium was a highly significant (P<0.0001) predictor of myocardial infarction in men, with an odds ratio of 1.2 per 0.1 mmol/L increase in serum calcium. In women, a nonsignificant trend was again seen. Because the free or ionized form of calcium is the physiologically important form and serum calcium was not corrected for serum albumin in our study, the results must be interpreted with caution. However, it appears likely that serum calcium is a predictor of cardiovascular disease in men.     

Effects of copper-aspirin complex on plasma 6-keto-prostaglandin F1α level and platelet cytosolic calcium in rabbits

Effects of copper-aspirin complex on washed platelet aggregation, thromboxane B₂ formation and 6-ketoprostaglandin F_1α level were monitored by Born's and Terashita's methods, respectively. The influence of copper-aspirin complex on cytosolic free calcium was examined using the fluorescent indicator, Fura 2-AM. Copper-aspirin complex significantly inhibited arachidonic acid-induced aggregation in washed platelets. The IC₅₀ value was 9.6 μmolL^-1. Copper-aspirin complex significantly decreased arachidonic acid-induced thromboxane B₂ formation by 87.1% in washed platelets. Ten mg kg^-1 of copper-aspirin complex given intragastrically markedly increased the plasma level of 6-keto-prostaglandin F_1α . Aspirin, however, reduced both thromboxane B₂ formation and 6-keto-prostaglandin F_1α level. In the presence of CaCl₂ 1 mmolL^-1, copper-aspirin complex (20, 40 and 80 mu mol L^-1) markedly lowered arachidonic acid-induced increase in platelet calcium from the resting level (270±36 nmolL^-1) to 213±14, 170±20 and 135±17 nmolL^-1, respectively. In the presence of ethylene glycol-bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid 1 mmolL^-1, copper-aspirin complex (20, 40 and 80 mu molL^-1) significantly suppressed the release of intracellular calcium induced by arachidonic acid from 127±23 nmolL^-1 to 108±17, 93±12 and 70±13 nmolL^-1.