Drugs for the treatment of hyperlipidemia.

Hyperlipidemia is a major risk factor for the development of cardiovascular disease. Considerable evidence suggests that drugs with the ability to lower low density lipoprotein-cholesterol also lower cardiovascular deaths. This review will focus on new developments in the design of pharmacological agents and therapies that can favorably affect hyperlipidemia. A broad range of therapeutic agents will be discussed, including food additives, nutraceuticals, conventional pharmaceuticals as well as gene therapy. The potential adverse effects caused by these agents will also be discussed. Appetite suppressants, intestinal lipase inhibitors, inhibitors of dietary cholesterol absorption, inhibitors of chylomicron assembly, HMG CoA reductase inhibitors (statins) and adenovirus-mediated gene therapy are specifically discussed in detail. Treatments and issues specific to the elderly and to children are also described. The specific application of pharmacogenics to the problem of hyperlipidemia is presented as a likely direction for future research emphasis.

Nonmuscle myosin II localizes to the Z-lines and intercalated discs of cardiac muscle and to the Z-lines of skeletal muscle.

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh-frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with alpha-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.

Neonatal, lethal noncompaction of the left ventricular myocardium is allelic with Barth syndrome.

Loss-of-function mutations in the G4.5 gene have been shown to cause Barth syndrome (BTHS), an X-linked disorder characterized by cardiac and skeletal myopathy, short stature, and neutropenia. We recently reported a family with a severe X-linked cardiomyopathy described as isolated noncompaction of the left ventricular myocardium (INVM). Other findings associated with BTHS (skeletal myopathy, neutropenia, growth retardation, elevated urinary organic acids, and mitochondrial abnormalities) were either absent or inconsistent. A linkage study of the X chromosome localized INVM to the Xq28 region near the BTHS locus, suggesting that these disorders are allelic. We screened the G4.5 gene for mutations in this family with SSCP and direct sequencing and found a novel glycine-to-arginine substitution at position 197. This position is conserved in a homologous Caenorhabditis elegans protein. We conclude that INVM is a severe allelic variant of BTHS with a specific effect on the heart. This finding provides further structure-function information about the G4.5 gene product and has implications for unexplained cases of severe infantile hypertrophic cardiomyopathy in males.

Xq28-linked noncompaction of the left ventricular myocardium: prenatal diagnosis and pathologic analysis of affected individuals.

Isolated noncompaction of the left ventricular myocardium (INVM) is characterized by the presence of numerous prominent trabeculations and deep intertrabecular recesses within the left ventricle, sometimes also affecting the right ventricle and interventricular septum. Familial occurrence of this disorder was described previously. We present a family in which 6 affected individuals demonstrated X-linked recessive inheritance of this trait. Affected relatives presented postnatally with left ventricular failure and arrhythmias, associated with the pathognomonic echocardiographic findings of INVM. The usual findings of Barth syndrome (neutropenia, growth retardation, elevated urinary organic acids, low carnitine levels, and mitochondrial abnormalities) were either absent or found inconsistently. Fetal echocardiograms obtained between 24-30 weeks of gestation in 3 of the affected males showed a dilated left ventricle in one heart, but were not otherwise diagnostic of INVM in any of the cases. Four of the affected individuals died during infancy, one is in cardiac failure at age 8 months, and one is alive following cardiac transplant at age 9 months. The hearts from infants who died or underwent transplantation appeared, on gross examination, to be enlarged, with coarse, deep ventricular trabeculations and prominent endocardial fibroelastosis. Histologically, there were loosely organized fascicles of myocytes in subepicardial and midmyocardial zones of both ventricles, and the myocytes showed thin, often angulated fibers with prominent central clearing and reduced numbers of filaments. Markedly elongated mitochondria were present in some ventricular myocytes from one specimen, but this finding was not reproducible. Genetic linkage analysis has localized INVM to the Xq28 region, where other myopathies with cardiac involvement have been located.

Contribution of the sodium-calcium exchanger to contractions in immature rabbit ventricular myocytes.

In immature cardiac myocytes, the sarcoplasmic reticulum is sparse. Thus, we hypothesized that sarcolemmal Ca2+ influx through Na(+)-Ca2+ exchange is the dominant mechanism for modulating intracellular Ca2+ during contractions in fetal and neonatal hearts. We measured Na(+)-Ca2+ exchange currents in neonatal and adult rabbit ventricular cells using a rapid solution switch into 0 mM external Na+. The current densities (mean +/- SEM) were larger in 8 neonatal cells than in 10 adult cells (5.4 +/- 1.38 versus 1.65 +/- 0.25 pA/pF). Intracellular Ca2+ transients after inhibiting the sarcoplasmic reticulum with ryanodine and thapsigargin were unchanged in 15 neonatal cells, but decreased in 15 adult cells to 78.9 +/- 5.6% of baseline. When the Ca2+ channels were also inhibited by adding nifedipine, Ca2+ transients from Na(+)-Ca2+ exchange were 30.0 +/- 3.5% of baseline in neonatal cells compared with 13.4 +/- 3.4% in adult cells. Simultaneous contractions were a larger percent of baseline in neonatal cells (85.7.6 +/- 6.4%) than in adult cells (78.9 +/- 5.6%) after inhibiting the sarcoplasmic reticulum, and were unmeasureable in many cells from both age groups after inhibiting the Ca2+ channels as well. The ratio of Na(+)-Ca2+ exchanger mRNA to sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels decreased from 1.0 +/- 0.13 to 0.4 +/- 0.03 to 0.26 +/- 0.02 in fetal, neonatal and adult ventricles, respectively. These measurements were consistent with a dominant role for the Na(+)-Ca2+ exchanger in the immature heart.

The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells.

We investigated the effect of exchanger inhibitory peptide (XIP) on Na-Ca exchange current (INa-Ca) in guinea pig ventricular cells. Cells were voltage-clamped with microelectrodes containing 20 mM Na+ and 14.0 mM EGTA ([Ca]i = 100 nM). An outward putative exchange current was stimulated when extracellular Na+ was reduced from 144 mM to zero (Li+ replaced Na+). This outward current showed a significant dependence on extracellular Ca2+. When Na+ removal was delayed for up to 40 minutes (in the absence of extracellular K+ or the presence of 3.0 mM ouabain to block the Na+ pump), outward INa-Ca increased presumably because [Na]i increased. Time-dependent increases of outward current in the absence of K+ could be abolished by reapplication of K+, which presumably reactivates the Na+ pump and reduces intracellular Na+. This effect is blocked in the presence of 3.0 mM ouabain. The dependence of this current on extracellular Ca2+, its dependence on intracellular Na+, and activation by extracellular Na+ reduction, together with its resistance to ouabain all suggest that it is a Na-Ca exchange current. After dialyzing the cell with 10 microM XIP, outward INa-Ca was largely abolished. This indicates that XIP, which is a rather large molecule, can enter the heart cell via the microelectrode in sufficient quantities to inhibit exchange. Inward INa-Ca was blocked secondary to the blockade of outward INa-Ca. L-type Ca2+ current (ICa) was not measurably affected by XIP.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in cardiac myocyte calcium regulation.

Developmental changes in the contributions of transsarcolemmal Ca2+ influx and Ca2+ release from intracellular storage sites to myocardial contraction were evaluated in isolated ventricular myocytes from neonatal (aged 1-7 days) and adult (aged 8-10 weeks) New Zealand White rabbits. Contractions ceased in one beat when extracellular Ca2+ was decreased from 1mM to micromolar levels using a rapid perfusion technique. On reperfusion with 1 mM Ca2+, recovery of control contraction amplitude occurred after significantly fewer beats in neonatal myocytes compared with adult myocytes, and after 1 minute compared with 5 minutes of reduced Ca2+. After 15 minutes of perfusion with either 1 or 10 microM ryanodine, contraction amplitude decreased in both age groups, but the decrease was significantly greater in adults than in neonates. These experiments indicate that isolated ventricular myocytes may be used in the study of developmental changes in intracellular Ca2+ regulation. Results suggest that cardiac contraction in neonates is relatively more dependent on transsarcolemmal Ca2+ influx. Furthermore, although Ca2+ release from intracellular storage sites is present in both neonates and adults, its role in cardiac contraction is more significant in adults.

Isolated noncompaction of left ventricular myocardium. A study of eight cases.

Isolated noncompaction of left ventricular myocardium is a rare disorder of endomyocardial morphogenesis characterized by numerous, excessively prominent ventricular trabeculations and deep intertrabecular recesses. This study comprised eight cases, including three at necropsy. Ages ranged from 11 months to 22.5 years, with follow-up as long as 5 years. Gross morphological severity ranged from moderately abnormal ventricular trabeculations to profoundly abnormal, loosely compacted trabeculations. Echocardiographic images were diagnostic and corresponded to the morphological appearances at necropsy. The depths of the intertrabecular recesses were assessed by a quantitative echocardiographic X-to-Y ratio and were significantly greater than in normal control subjects (p less than 0.001). Clinical manifestations of the disorder included depressed left ventricular systolic function in five patients, ventricular arrhythmias in five, systemic embolization in three, distinctive facial dysmorphism in three, and familial recurrence in four patients. We conclude that isolated noncompaction of left ventricular myocardium is a rare if not unique disorder with characteristic morphological features that can be identified by two-dimensional echocardiography. The incidence of cardiovascular complications is high. The disorder may be associated with facial dysmorphism and familial recurrence.

Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH.

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.

Alzheimer's paired helical filaments share epitopes with neurofilament side arms.

A panel of monoclonal antibodies to neurofilaments have been investigated with regard to the location of their respective epitopes on neurofilament polypeptides and their ability to label the neurofibrillary tangles and paired helical filaments (PHF) which are characteristic of Alzheimer's disease. All of the neurofilament monoclonal antibodies that label tangles and PHF are directed against epitopes in the side arm domains of the two larger neurofilament polypeptides, NF-H and NF-M, and do not recognise the alpha-helical rod domains of these proteins. Immuno-electron microscopy demonstrates that the neurofilament antibodies label the constituent PHF per se and do not simply stain neurofilaments that might be admixed with PHF. These neurofilament epitopes are differentially retained by PHF, following isolation. Thus, antibody labelling of PHF is not simply due to the presence of normal neurofilament polypeptides. We propose that in tangle-bearing neurons, neurofilaments are degraded by proteases and that it is fragments of the side arms which contribute to the composition of PHF.

In vitro development of third- and fourth-stage larvae of Dirofilaria immitis: comparison of basal culture media, serum levels and possible serum substitutes.

In vitro development and survival of third-stage larvae of Dirofilaria immitis were compared in four different culture media and in the presence of varying concentrations of four different medium supplements. Motility and the incidence of third- to fourth-stage molting were used as criteria for evaluating different culture conditions. No significant differences in either motility or molting response were detected between larvae cultured in NCTC-135, F12(K), CMRL 1066 or Dulbecco's Modified Eagle's Medium. Fetal calf serum enhanced development and survival of the cultured larvae in dose-dependent fashion. Its effects were maximal at a concentration of 20 percent of the total medium volume. Addition of a commercial medium supplement, NuSerum, also gave a dose-related increase in larval development and viability. The activity of NuSerum in this respect was comparable to that of fetal calf serum. The tripeptide glycylhistidyllysine and bovine serum albumin, fraction V both failed to stimulate development of third-stage D. immitis larvae in vitro.

The proteolytic digestion of ox neurofilaments with trypsin and alpha-chymotrypsin.

Brief digestion of ox neurofilaments with trypsin liberates fragments that are soluble and have molecular weights ranging from 164 000 to 97 000. Peptide fingerprinting indicates that these regions, termed the tryptic head-regions, arise from the 205 000- and 158 000-mol.wt. components of the triplet. The remains of the parent polypeptides sediment with normal filaments and have been termed tail-regions. Digestion of neurofilaments with chymotrypsin also liberates soluble fragments (chymotryptic head-regions) but these have mol.wts. 171 000 and 119 000, though they too originate from the higher-molecular-weight triplet polypeptides. Tryptic and chymotryptic head-regions have extensive homology, and a low (less than or equal to 20%) helix content. Electron microscopy shows that chymotryptic digestion rapidly reduces the length of filaments, probably because this enzyme preferentially attacks the 72 000-mol.wt. polypeptide. In contrast, brief digestion with trypsin does not reduce filament length even though more than 90% of the two higher-molecular-weight components have been cleaved. These results indicate that the backbone of native filaments is formed from the 72 000-mol.wt. polypeptide together with the tail-regions from the 205 000- and 158 000-mol.wt. polypeptides. The corresponding head-regions of these components, which can represent nearly 75% of each molecule, are not necessary for preserving the backbone of native neurofilaments and are therefore good candidates for being the side arms that connect these filaments in nerve cells.