Marcus A. Rothschild

Alcoholic cardiomyopathy II. The inhibition of cardiac microsomal protein synthesis by acetaldehyde

Abstract Previous studies in this laboratory had shown that while ethanol at levels of 200 to 300 mg 100 ml had no effect on cardiac protein synthesis, acetaldehyde ( 3.5 mg 100 ml or 0.8 m m ) markedly inhibited cardiac protein synthesis in the intact heart in vitro. In order to localize further the action of acetaldehyde and to separate the protein synthetic effects from contractile function, studies on cell free systems with cardiac muscle microsomes were carried out at concentrations of acetaldehyde seen in humans after moderate ethanol ingestion. There was a significant reduction of microsomal protein synthesis even at these levels of acetaldehyde. Thus, with an acetaldehyde concentration of 0.53 mg 100 ml (0.12 m m ) the protein synthesis was reduced to 52 ± 5.3% of the control microsomes. With acetaldehyde concentrations of 0.13 to 0.26 mg 100 ml (0.03 to 0.06 m m ), the microsomal protein synthesis was 65 ± 8.6% of the controls. The differences from the controls were statistically significant. These data show that at concentrations seen in humans following ethanol ingestion, acetaldehyde interferes with normal cardiac protein synthesis independent of contractile action and thus may play a role in the ultimate development of ethanolic cardiomyopathy.

The Stimulation of Albumin Synthesis by Methadone

Elevated levels of serum albumin have been noted in patients on chronic methadone maintenance and in heroin addicts. This observation was investigated in rabbits maintained on daily methadone 4 mg per kg of body weight after a period of 3 months on increasing dosage to assure drug tolerance. Albumin distribution and metabolism were measured with tested lots of 125I rabbit albumin. Studies were made before and again after the attainment of the methadone maintenance state. Albumin distribution was altered markedly with a shift of intravascular albumin to extravascular sites. Associated with this change, the serum albumin level rose by an average of 0.5 g per 100 ml. Albumin degradation increased by 32% from 248 to 327 mg per kg per day. The total exchangeable albumin pool increased 35%, or 3.6 g. Since the exchangeable albumin pool increased in the face of an increment in albumin degradation, albumin synthesis must have increased even further to account for this change. Although the specific factors responsible for these alterations in albumin metabolism and distribution are not known at present, to date, this hyperalbuminemic hypercatabolic state is not produceable in any other clinical or experimental situation.

Effects of ethanol on protein synthesis.

Cardiac: Cardiac protein synthesis is influenced by the state of nutrition with reduction of cardiac size in starvation. Ethanol per se may not affect this synthesis directly, but the metabolite of ethanol, acetaldehyde, profoundly decreases normal protein synthesis in the heart in vitro. The interference with the synthetic process may play a role in the ultimate cardiomyopathies of malnutrition and alcoholism. Hepatic: In vivo albumin synthesis is sensitive to environment, oncotic pressure, normal balance, nutrition, as well as toxins and state of health. Thus, to study the acute effects of alcohol alone, it was necessary to employ the isolated perfused liver. Fasting reduced albumin synthesis 50%, with loss of RNA and a disaggregation of the endoplasmic membrane bound polysome. Tryptophan, arginine and ornithine added to the perfusate at a final concentration of 10 mM reversed these findings. Alcohol likewise reduced albumin synthesis; disaggregates the bound polysome without a marked loss of RNA. Ornithine, arginine and tryptophan are able to reverse this loss in albumin synthesizing capacity. The combination of fasting and alcohol, while not lowering albumin synthesis below that seen with either stress alone, prevents the recovery from either stress.

Anginal Syndrome Induced by Gradual General Anoxemia

With individuals subject to attacks of chest pain, in whom there is no evidence by physical or electrocardiographic examination of myocardial (coronary) disease, it is often difficult to be certain of the origin of the pain. We wished to distinguish between those with pain due to impaired coronary circulation, and those in whom the pain arose otherwise. It occurred to us that if one were to produce a general anoxemia, and, therefore a local cardiac anoxemia, there might appear differentiating responses in these 2 groups. By means of rebreathing, we were able to produce a state of general anoxemia in human subjects. The carbon dioxide was absorbed. It usually took about 10 minutes for the oxygen to become so low that the patient became uncomfortable. Twenty-seven patients were subjected to the test. Fourteen patients were used as controls. The controls consisted of 4 patients with normal hearts. The remaining cases were patients with chronic valvular disease, paroxysmal auricular fibrillation, rheumatic fever, spondylitis, gall bladder disease, and cardiospasm. None of these patients developed pain. Thirteen were patients with clear-cut histories of attacks of precordial pain brought on by exertion, excitement, eating, or exposure to cold. Ten developed pain during the rebreathing test. Eight of these had no physical or electrocardiographic signs of myocardial disease. The pain appeared when the oxygen fell to about 9 to 10%. This ordinarily took about 8 to 10 minutes. Subjects were advised to raise their hands when they felt uncomfortable, and the experiment was stopped. It is furthermore interesting to observe that 2 other patients with clinical angina and intra-ventricular block developed pain and additional electrocardiographic changes during the anoxemia.

Increased cardiac contractility in high calcium perfusion and protein synthesis.

Cardiac stress produced by pressure overload results in increased protein synthesis, but it has not been possible to separate the effects of increased contractile action, per se, from those of increased pressure. In a model, where coronary flow may be controlled, right ventricular contractility, measured by dPdt max and ΔP (systolic minus diastolic pressures) was ascertained during perfusion with varied calcium concentrations while the pulmonary artery pressure was kept constant at control levels. In high calcium (4.8 m m CaCl2) dP/dt max was 165 compared to 51 mmHg. s−1 in controls (1.2 m m CaCl2) with the ΔP nearly doubled in the high calcium perfusion. Although these parameters of contractility increased in the high calcium perfusion to the same degree as with pressure overload in this model, protein synthesis was the same as in the controls with incorporation of [14C]lysine 30.9 μmol. g−1 protein N in the high calcium group and 31.1 μmol in the controls. The high calcium group also showed increased protein N and higher ATP levels in the face of unchanged water content or extracellular spaces. The results suggest that neither high calcium perfusion nor increased contractility per se are stimuli for increased cardiac protein synthesis.

Problems in evaluating cardiac protein synthesis

Abstract How significant are present measurements of cardiac protein synthesis? In the heart, alterations in protein synthesis have been reported to accompany changes in organ size and gross structure, but the mechanisms involved in altering the synthesis and the significance of these alterations in heart muscle in adaptation to stress (defined as an influence of an adverse force or strain) have not been easily clarified. The determination of cardiac protein synthesis presents many problems some of which are inherent in the methodologies used and others in the complexities of the protein synthetic process itself, and examination of these problems is necessary in any evaluation of synthesis.

Effects of Ethanol on Protein Synthesisa

Alcohol is known to affect protein synthesis and/or protein secretion.’-’’ In the case of hepatic-made proteins destined for secretion as extracellular proteins there is conflicting evidence as to which aspect is affected, synthesis or secretion, since these extracellular proteins are the net result of both processe~.’”~~ In the case of intracellular proteins secretion is not a factor. However, both intracellular proteins and those destined for export share common pathways of synthesis and intracellular transport. Protein synthesis begins in the nucleus with: 1) gene transcription into mRNA; 2) post-transcriptional processing of the mRNA including the excission of nucleotides, the addition of a polyadenylate sequence, and the formation of 5’ methylated termini (“cap”); and, finally, 3) secretion from the nucleus into the cytoplasm. It is in the cytoplasm that de facto protein synthesis occurs. Here, the small ribosomal subunit, which contains some initiation factors, forms a complex with the initiator tRNA and several other factors to form a pre-initiation complex. This is followed sequentially by the binding of mRNA and the large ribosomal subunit to form the active 80s initiation complex. Chain elongation occurs as the ribosome moves along the mRNA one codon a t a time. At each codon specific tRNAs with the appropriate anticodons bind to the mRNA, and the amino acid they carry is covalently linked to the growing polypeptide chain. Chain elongation continues until a termination codon is reached. Each step requires several protein factors as well as energy.24 In most types of eukaryotic cells there are two major classes of ribosomes: bound ribosomes and free ribosomes. The bound ribosomes are bound to an internal network of lipoprotein membranes-the endoplasmic reticulum. There is no structural difference between a free and a bound ribosome. Binding to the membrane occurs after protein synthesis has started, probably by means of a leader polypeptide chain (“signal hyp~thesis”).~’ This hypothesis can be satisfied by a number of possible mechanisms: binding to a receptor, or insertion across the lipid bilayer without another membraneprotein participation. Whether a ribosome binds or does not bind to the membrane depends upon the protein synthesized. Most proteins destined to be secreted or to be stored in intracellular vesicles are synthesized by ribosomes attached to the endoplasmic reticulum. These proteins are

Ion Diffusion Delay in the Beating Mammalian Heart.

Summary Rates of washout of Na24+ and I131were measured in the working perfused guinea pig heart in vitro at 37° and 25°C. Washout occurred at 2 or more rates with at least 80% of the ions comprising the fast component and exchanging at a mean rate of 1.03 min-1. There were no significant differences between the washout rates of the Na24+ and I131- at the 2 temperatures studied. Washout of tissue slices 400-500 μ thick occurred at a single rate one-tenth that of the fast component in the working perfused heart. The data indicate that ion diffusion delay in passing through the extracellular spaces does not play a significant role in evaluation of K transfer in this preparation, and they also support previous findings that the fast component of K exchange does not represent extracellular localization.

The determination of free ε-amino groups in proteins☆

Abstract 1. 1. p -Iodobenzene [ 35 S]sulfonyl (pipsyl) chloride has been used to determine the free e-aminos group in proteins by means of the isotope derivative-carrier method. 2. 2. Chromatography on A.R. Dowex-50 X4 citrate was used to isolate and quantitatively determine e - N -pipsyllysine and e - N -pipsylhydroxylysine. 3. 3. Results obtained with a number of well characterized proteins, including ichthyocol and spiny dogfish collagen have shown that all the e-amino groups were derivatized, and rules out their participation in interchain cross links.