I. T. Mak

Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation.

Sarcolemmal and microsomal membranes prepared from adult canine cardiac my-ocytes (sarcolemmal Na+,K+-ATPase = 71.8 /imol/mg per hr and microsomal rotenone-insensitive NADH cytochrome c reductase =114 μmol/mg per hr) were each preincubated at 37°C in the presence of a free radical generating system consisting of dihydroxyfumarate and Fe+++-ADP; loss of the Na+,K+-ATPase and reductase activities, as well as the associated increases in lipid peroxidation, measured by malondialdehyde formation, were temporally correlated in both systems. The ATPase was inhibited 70% when the malondialdehyde was 71 nmol/mg protein at 20 minutes and 90% when malondialdehyde was 138 nmol/mg protein at 90 minutes. Inhibition of reductase activity occurred more gradually, displaying a 27% loss of activity when malondialdehyde reached 34 nmol/mg protein at 20 minutes and 60% with a malondialdehyde value of 67 nmol/mg protein at 90 minutes. The greater susceptibility of the sarcolemma to free radical-induced membrane damage may be due to the higher content of unsaturated fatty acids in this membrane, compared to microsomes.

Role of free radicals and substance P in magnesium deficiency

In the United States the literature contains only sporadic references to clinical disorders of Mg-deficiency, compared to more recent interest in the benefits of magnesium infusion in myocardial infarction and other acute clinical conditions [1,2]. In Europe the clinical interest in Mg-deficiency was pioneered by Durlach in his book entitled Le Magnesium en Prutique Clinique; the English edition was entitled Magnesium in Clinical Practice [3]. In the conclusion of his book, Durlach stated: “This ion which is present in all the cells is involved in many different pathologies. Integrating a search for the disorders of magnesium metabolism in daily diagnostic processes allows determination of the indications and precise methods of magnesium therapy.” In the United States, Seelig authored a text in 1980 entitled Magnesium Dejicicncy in the Parhogerzesis sf Disease [4] and reviewed the literature concerning magnesium requirements in human nutrition and the association of magnesium deficiency with cardiovascular disease [s]. That same year Wacker published an excellent book entitled Mugnesium und Mm in which he emphasized the clinical relevance of magnesium 161. Six decades ago MacCollum [7l studied the effects of Mg-deficiency on development, reproduction, neuromuscular and humoral abnormalities in animals. In 1959 Bajusz and Selye published a paper describing the influence of electrolytes in the process of myocardial injury [8]. More recently, Lehr focused attention on magnesium and the process of cardiac necrosis [9], or cardiomyopathic lesions which had been described earlier [lo]. B.T. Altura and B.M. Altura published a series of papers which postulated

Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: Effects of substance P-receptor inhibition

Dietary deficiency of magnesium (Mg) in rodents results in cardiomyopathic lesion formation. In our rat model, these lesions develop after 3 weeks on the Mg-deficient diet; significant elevation of several cytokines, IL-1, IL-6 and TNFα also occurs. In probing the mechanisms of lesion formation, we obtained data supporting the participation of free radicals (Freedman AMet al.: Bioch Biophys Res Commun 1990; 170: 1102). Recently, we identified an early elevation of circulating substance P and proposed a role of neurogenic peptides during Mg-deficiency (Weglicki WB, Phillips TM: Am J Phys 1992; 262: R734). The present study was designed to evaluate the contribution of neurogenic peptides to the pathogenesis of Mg-deficiency. In the blood, substance-P and calcitonin gene related peptide (CGRP) are elevated during the first week on the diet. During the second week, circulating histamine, PGE2 and TBAR-materials were elevated and red cell glutathione was reduced, all prior to the elevation of the inflammatory cytokines during the third week. When the rats were treated with the substance P-receptor blocker [CP-96,345], the levels of substance P and CGRP remained elevated; however, increases in histamine, PGE2, TBAR-materials, and the decrease in red cell glutathione were inhibited; also, the development of cardiac lesions was inhibited significantly. These data support a central role for neurogenic peptides, especially substance P, in the development of cardiomyopathic lesions during Mg-deficiency.

Blockade of cardiac inflammation in Mg2+ deficiency by substance P receptor inhibition.

In previous work we reported the elevation of circulating inflammatory cytokines in rodents maintained on a Mg(2+)-deficient diet. Within the first week of Mg2+ deficiency, significant elevation of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) occurs. The present study was designed to assess the effects of SP receptor blockade by CP-96,945 and its inactive enantiomer CP-96,344 on tissue cytokine levels and in vivo oxidative indexes. CP-96,345 had no significant effect on circulating levels of SP or CGRP; however, at the tissue level, a significant decrease (P < .01) in myocardial accumulation of SP occurred; the inactive enantiomer was only slightly effective. In addition, CP-96,345 significantly reduced (by 53%) the accumulation of tumor necrosis factor-alpha (TNF-alpha) (but not interleukin-1 and interleukin-6) within the lesions; the effect of the enantiomer was insignificant. We conclude that treatment with CP-96,345 inhibits SP and TNF-alpha tissue levels in cardiac lesions, indicating a linkage between this neuropeptide and TNF-alpha. Both SP and TNF-alpha can trigger free radical production; plasma thiobarbituric acid-reactive materials were elevated 2.5-fold and red blood cell reduced glutathione was reduced 55% during Mg2+ deficiency. In the presence of CP-96,345, both indexes of in vivo oxidation were significantly attenuated; the enantiomer was ineffective. These latter observations point to a neuropeptide/TNF-alpha/free radical-triggered mechanism that may be the major pathway of systemic oxidative injury inducing the cardiomyopathic lesions seen during Mg2+ deficiency.

The nerve-heart connection in the pro-oxidant response to Mg-deficiency.

Magnesium is a micronutrient essential for the normal functioning of the cardiovascular system, and Mg deficiency (MgD) is frequently associated in the clinical setting with chronic pathologies such as CHF, diabetes, hypertension, and other pathologies. Animal models of MgD have demonstrated a systemic pro-inflammatory/pro-oxidant state, involving multiple tissues/organs including neuronal, hematopoietic, cardiovascular, and gastrointestinal systems; during later stages of MgD, a cardiomyopathy develops which may result from a cascade of inflammatory events. In rodent models of dietary MgD, a significant rise in circulating levels of proinflammatory neuropeptides such as substance P (SP) and calcitonin gene-related peptide among others, was observed within days (1–7) of initiating the Mg-restricted diet, and implicated a neurogenic trigger for the subsequent inflammatory events; this early “neurogenic inflammation” phase may be mediated in part, by the Mg-gated N-methyl-D-aspartate (NMDA) receptor/channel complex. Deregulation of the NMDA receptor may trigger the abrupt release of neuronal SP from the sensory-motor C-fibers to promote the subsequent pro-inflammatory changes: elevations in circulating inflammatory cells, inflammatory cytokines, histamine, and PGE2 levels, as well as formation of nitric oxide, reactive oxygen species, lipid peroxidation products, and depletion of key endogenous antioxidants. Concurrent elevations of tissue CD14, a high affinity receptor for lipopolyssacharide, suggest that intestinal permeability may be compromised leading to endotoxemia. If exposure to these early (1–3 weeks MgD) inflammatory/pro-oxidant events becomes prolonged, this might lead to impaired cardiac function, and when co-existing with other pathologies, may enhance the risk of developing chronic heart failure.

The role of magnesium deficiency in cardiovascular and intestinal inflammation.

Hypomagnesemia continues to cause difficult clinical problems, such as significant cardiac arrhythmias where intravenous magnesium therapy can be lifesaving. Nutritional deficiency of magnesium may present with some subtle symptoms such as leg cramps and occasional palpitation. We have investigated dietary-induced magnesium deficiency in rodent models to assess the pathobiology associated with prolonged hypomagnesemia. We found that neuronal sources of the neuropeptide, substance P (SP), contributed to very early prooxidant/proinflammatory changes during Mg deficiency. This neurogenic inflammation is systemic in nature, affecting blood cells, cardiovascular, intestinal, and other tissues, leading to impaired cardiac contractility similar to that seen in patients with heart failure. We have used drugs that block the release of SP from neurons and SP-receptor blockers to prevent some of these pathobiological changes; whereas, blocking SP catabolism enhances inflammation. Our findings emphasize the essential role of this cation in preventing cardiomyopathic changes and intestinal inflammation in a well-studied animal model, and also implicate the need for more appreciation of the potential clinical relevance of optimal magnesium nutrition and therapy.

Inhibition of tumor necrosis factor-alpha by thalidomide in magnesium deficiency.

The effect of thalidomide on circulating cytokines and myocardial lesion formation was investigated in Mg-deficient rats. After two weeks on a Mg-deficient diet, rats show an increase in circulating levels of tumor necrosis factor-alpha and interleukin 1. Thalidomide (1 mg/day) caused a complete inhibition of the increase in circulating tumor necrosis factor-alpha levels, without having an effect on interleukin 1. However, a marked increase in cardiomyopathic lesion formation was observed in Mg-deficient animals treated with thalidomide; possible mechanisms for thalidomides enhancement of myocardial injury are discussed. (Mol Cell Biochem129: 195–200, 1993)

The Role of Antioxidant Drugs in Oxidative Injury of Cardiovascular Tissue

For more than a decade, our laboratory has investigated the signi~cance of oxygen derived-free radicals during myocardial ischemia/reperfusion in several animal [1–6] and clinical [7] models. We have also studied the antioxidant properties of cardiovascular drugs in vitro and in vivo and have assessed their ability to inhibit myocardial injury [8–18]. Over the years, other laboratories have corroborated our ~ndings. Animal studies and limited clinical observations have also lent support to the importance of antioxidant therapy for certain cardiovascular diseases including cardiomyopathy. By no means comprehensive, this communication serves as a brief review of these efforts for selected groups of pharmacological agents with antioxidant activities and their potential clinical relevance.

Cardiovascular Membranes as Models for the Study of Free Radical Injury

Following ischemia — a condition characterized by oxygen depletion, acidosis, and retention of tissue metabolites (1) — severe oxidative damage to cardiovascular tissues occurs upon reperfusion. However, early reperfusion remains the major clinical method to salvage ischemic myocardium and further elucidation of the multiple injury mechanisms operative during ischemia/reperfusion is necessary to solve this paradoxical dilemma. In the process of ischemia/reperfusion, mounting evidence has confirmed that the generation of oxygen-derived free radicals participates in the injury process (2–4). At the whole organ level, the reported protection of cardiovascular tissues by antioxidant-type enzymes (eg. superoxide dismutase and catalase), antioxidant drugs (eg. deferoxamine) and vitamins (eg. vitamin E) suggest a role for free radicals in the reperfusion injury. In recent years studies employing electron spin resonance spectroscopy and spin-trapping agents have shown that increased production of free radicals occurs during ischemia and myocardial reperfusion (4–11). The specific cellular sites of generation of these toxic free radicals are not known. Certainly white cells migrating into ischemic/reperfused areas of myocardium are capable of generating significant amounts of superoxide anions (2,12–14); endothelial cells (15–17) and cardiomyocytes (18) have been reported to generate oxygen-derived free radicals as well. Nevertheless, the relative contribution of each of the various cell types to the overall process of injury of cardiovascular tissues remains an area of intense investigation. One significant challenge for this area of investigation is whether or not sufficient radicals accumulate to overwhelm the intrinsic cellular protective mechanisms.