J. Zähringer

Regulation of atrial natriuretic factor release in man: Effect of water immersion

The atrial natriuretic factors (ANF) have recently emerged as a novel hormonal system [3, 10] that may be implicated in extracellular volume homeostasis [9]. Recently, employing our highly sensitive radioimmunoassay [1], we have demonstrated elevated ANF plasma levels in patients with diseases involving pressure/volume overload [2, 6]. The physiologic regulation of ANF release in healthy subjects, however, needs further elucidation [7]. Water immersion causes an increase in central venous pressure due to a shift of extracellular volume to the intrathoracic venous bed [4]. This increase in central venous and atrial pressure has been shown to increase diuresis and natriuresis [5, 8]. In the present study, the hypothesis is tested, that water immersion, via the increase in central venous pressure, stimulates an enhanced release of ANF into the circulation that may contribute to the ensuing diuresis.

The effect of triiodothyronine on the cardiac mRNA.

Abstract Cardiac hypertrophy was induced in rats by daily injections of L-triiodothyronine (0.2 mg/kg body wt, s.c., 1 to 3 days). Regression of hypertrophy was studied after 3 days of injection for a period of 1 to 16 days. Cardiac RNA was isolated by phenolextraction, mRNA by affinity chromatography of cardiac total RNA on oligo-dT-cellulose. In addition, cardiac mRNA was also quantitated by hybridization to 3 H-Poly(U). After 3 days of T 3 -treatment, the extractable amount of cardiac total RNA increased from 759 ± 181 μg/g heart (controls) to 980 ± 119 μg/g heart (T 3 -rats), and that of mRNA from 24.7 ± 2.0 (controls) to 39.4 ± 7.0 (T 3 -rats), increases of 29.1% (total RNA) and 59.5% (mRNA). 16 days after cessation of T 3 -treatment, total RNA and mRNA levels had returned to normal. The increase in cardiac mRNA in T 3 -rats was investigated in further detail by in vitro translation of the isolated mRNA in the mRNA-dependent reticulocyte lysate system. Incorporation of 35 S-methionine into total protein was identical for cardiac mRNA from control and T 3 -rats. Separation of the in vitro synthesized proteins on SDS-Polyacrylamide-Slabgels revealed the complete synthesis of actin, troponin-T, tropomyosin, myoglobin and myosin-light-chains 1 and 2. The mRNAs coding for these proteins represented identical fractions of total mRNA in control and T 3 -rats. This leads to the conclusion that T 3 had stimulated the synthesis of all of these specific mRNAs in an unselective manner. From our data and from the results of other authors we conclude that changes in cardiac mRNA levels are the major regulatory factor in causing changes in cardiac protein synthesis rates leading to cardiac hypertrophy in hyperthyroidism.

The regulation of protein synthesis in heart muscle under normal conditions and in the adriamycin-cardiomyopathy

SummaryThe regulation of cardiac protein synthesis, in particular messenger-RNA (mRNA) and polyribosome metabolism, has been investigated in normal rat heart muscle and in the adriamycin-cardiomyopathy by using newly developed methods for the isolation, characterization and in-vitro translation of cardiac polyribosomes and mRNA. The obtained data allow the following conclusions:1.Normal heart muscle has a high content of polyribosomes (865 µg/g) and of mRNA (20–60 µg/g), and thus a high rate of protein synthesis.2.The level of cardiac polyribosomes and mRNA is strictly age-dependent and much higher in young animals (2–3 ×). This corresponds to a higher cardiac protein synthesis rate in young animals with a growing heart muscle, and shows that the protein-synthetic reserves of heart muscle decrease sharply with age.3.Withdrawal of food for 1–3 days results in a pronounced decrease (−50% to −70%) of cardiac polyribosomes and mRNA, demonstrating that the cardiac protein synthesis reacts very sensitively to conditions of starving.4.The cardiac polyribosomes and mRNA are unevenly distributed in the myocyte. The bulk of these substances is present in the cardiac microsomes, and much less is found in nuclei, myofibrils, mitochondria and in the post-microsomal fraction (=cell-sap) of the cardiac muscle. This shows that the major intracellular site of cardiac protein synthesis is the microsomal fraction of the myocyte.5.A pool of untranslated mRNA was demonstrated to be present in the cell-sap of the myocyte. This mRNA is to some extent translatable in-vitro and appears to represent mRNA sub-pools with two functions: a) mRNA which is partially broken down or in the process of being broken down, and b) intact mRNA which could have a “reserve-function”, e.g., by being utilized to increase cardiac protein synthesis under certain conditions.6.A method of quantitating small amounts of cardiac mRNA (25–50 ng) has been developed which makes it possible to estimate the mRNA content of cardiac biopsies.7.These methods were utilized to study the relevance of changes in RNA- and protein synthesis in the development of the adriamycin-cardiomyopathy. It appears that severe decreases in cardiac mRNA and polyribosome levels are a key factor in the pathogenesis of the adriamycin-cardiomyopathy. These decreases are probably caused by the direct binding of adriamycin to cardiac DNA and lead themselves to a persisting decrease in cardiac protein synthesis which in view of the short half-lives of the cardiac contractile proteins (5–12 days) causes a gradual loss of cardiac structure and function.ZusammenfassungDie Regulation der myokardialen Proteinsynthese, insbesondere der Messenger-RNS (mRNS) und Polyribosomen Metabolismus, wurden in normalem Rattenherzmuskel und in der Adriamycin-Kardiomyopathie untersucht. Dabei wurden neu-entwickelte Methoden zur Isolierung, Charakterisierung und in-vitro Translation myokardialer Polyribosomen und mRNS eingesetzt. Die erhaltenen Ergebnisse erlauben folgende Folgerungen:1.Normaler Herzmuskel hat einen hohen Gehalt an Polyribosomen und mRNS und daher eine hohe Proteinsyntheserate.2.Der Gehalt des Herzmuskels an Polyribosomen und mRNS ist stark altersabhängig und wesentlich höher bei jungen Tieren (2–3×). Dies entspricht der höheren Proteinsyntheserate des wachsenden Herzmuskels junger Tiere und zeigt, daß die Proteinsynthesereserven des Herzmuskels mit steigendem Alter stark abnehmen.3.Nahrungsentzug für 1–3 Tage führt zu einer ausgeprägten Abnahme (−50% bis −70%) der myokardialen Polyribosomen und mRNS und unterstreicht die Empfindlichkeit der Herzmuskel-Proteinsynthese hinsichtlich auch nur kurzer Hungerperioden.4.Die intrazelluläre Verteilung der myokardialen Polyribosomen und mRNS ist sehr ungleichmäßig: Die große Mehrzahl dieser Substanzen befindet sich in der Mikrosomenfraktion des Herzmuskels; Zellkerne, Myofibrillen, Mitochondrien und der post-mikrosomale Überstand (=Zellsaft) enthalten nur jeweils geringe Mengen. Dies zeigt, daß der intrazelluläre Hauptort der myokardialen Proteinsynthese in der Mikrosomenfraktion des Herzmuskels liegt.5.Ein Pool nicht-translatierter mRNS wurde von uns im Zellsaft des Herzmuskels nachgewiesen. Diese mRNS kann in-vitro teilweise translatiert werden und scheint sich aus zwei Fraktionen zusammenzusetzen: a) mRNS, die gerade abgebaut wird oder schon abgebaut ist, und b) intakte mRNS, die eine „Reserve-Funktion“ wahrnehmen könnte, z.B. durch Stimulierung der myokardialen Proteinsynthese unter bestimmten Bedingungen.6.Durch eine neu-entwickelte Methode kann myokardiale mRNS in geringen Mengen (25–50 ng) quantifiziert werden. Dies eröffnet die Möglichkeit, den mRNS-Gehalt von Herzmuskelbiopsien zu bestimmen.7.Diese Methoden wurden zur Überprüfung der Frage eingesetzt, inwieweit Veränderungen der RNS-und Proteinsynthese bei der Entstehung der Adriamycin-Kardiomyopathie eine Rolle spielen könnten. Unsere Daten lassen den Schluß zu, daß ausgeprägte Abnahmen der myokardialen mRNS- und Polyribosomenspiegel eine entscheidende Rolle bei der Pathogenese der Adriamycin-Kardiomyopathie spielen. Diese Abnahmen sind wahrscheinlich durch eine direkte Bindung von Adriamycin an die myokardiale DNS bedingt und führen ihrerseits zu einer persistierenden Abnahme der myokardialen Proteinsynthese, die aufgrund der kurzen Halbwertszeit der myokardialen Proteine (5–12 Tage) einen allmählichen Verlust der strukturellen und funktionellen Integrität des Myokards zur Folge hat.

Virus myocarditis: Molecular hybridization allows the detection of virus-RNA in heart muscle after virus infection

In most patients with virus myocarditis, the diagnosis is still based on clinical data alone. Endomyocardial biopsies subjected to electron microscopy, immunofluorescence techniques and virus isolation procedures provide additional, but only occasionally conclusive information. In this communication we describe a new method which could possibly be used to improve the diagnostic possibilities in patients with suspected virus myocarditis. The method is based on the hybridization of radioactive complementary nucleotide sequences to virus-RNA. It is shown that in an experimental model (reovirus infected baby mice) this method can be used to demonstrate the virus infection of cardiac muscle. It is suggested that the method could be adapted to other viruses (e.g. coxsackie virus) and to endomyocardial biopsies derived from patients with suspected virus myocarditis.

Influence of starvation and total protein deprivation on cardiac mRNA levels.

SummaryThe effect of starvation and of protein-deprivation on the extractable amount of cardiac mRNA was investigated in male rats. Cardiac mRNA was determined by either (a) isolations of cardiac mRNA by SDS-Phenol/oligo-dT-cellulose, or by (b) hybridization of cardiac mRNA to3H-Poly(U). During starvation (1–6 days) the extractable amount of cardiac microsomal RNA decreased from 870 μg/g heart (controls) to 606 μg/g (3 days) and to 547 μg/g (6 days), the extractable amount of mRNA fell from 28.6 μg/g heart (controls) to 18.7 μg/g (3 days) and to 14.5 μg/g (6 days). When a normocaloric but proteindeficient diet was fed, the decreases in cardiac microsomal RNA and mRNA were qualitatively similar, out slightly less severe. An analysis of the intracellular distribution of cardiac microsomal RNA and mRNA in the hearts of normal animals and of animals starved or fed a protein-deficient diet indicates that during starvation cardiac mRNA does not accumulate in the cell sap, but gets rapidly degraded. In the refeeding period, mRNA is transported from the nucleus to the cytoplasm and engages in polyribosome formation. The specific mRNA species coding for the major myofibrillar cardiac proteins are affected to a similar extent by these changes during starvation/protein-deprivation and refeeding.

Isolation and characterization of structurally and functionally intact polyribosomes and mRNA from rat heart muscle

Optimal conditions for the isolation and in vitro translation of structurally and functionally intact polyribosomes and poly(A)-containing messenger-RNA (mRNA) from adult rat heart muscle are described. Using these conditions, 1 g heart muscle (wet weight) yields 865 μg polyribosomes, 719 μg microsomal RNA and 17.7 μg poly(A)-containing mRNA. The isolated polyribosomes, microsomal RNA and mRNA were structurally characterized by sedimentation analysis on continuous, 10 to 40% sucrose gradients (polyribosomes) and 5 to 40% sucrose gradients (microsomal RNA and mRNA). The polyribosomal profiles showed most of the polyribosomes sedimentating as heavy polyribosomes in the high S-value range. Microsomal RNA sedimentated in three distinct peaks corresponding to 4 S (tRNA), 18 S (rRNA) and 28 S (rRNA). The mRNA showed a heterogeneous sedimentation behaviour with a broad peak at about 20 S. Evidence for the functional intactness of the isolated polyribosomes and mRNA was obtained by their excellent capacities for in vitro protein synthesis in cell-free systems. The cell-free synthesized proteins were identified by analysis on SDS-polyacrylamide-slab gels and subsequent fluorography. Major radioactivity bands are found in the region of actin, troponin-T, tropomyosin, L1 and L2-myosin and myoglobin.

[The regulation of protein synthesis in heart muscle. Biochemical data, stimulative and inhibitory factors and their clinical significance (author's transl)].

The regulation of protein synthesis in heart muscle has been investigated by many authors under both normal and pathological conditions. This review summarizes the evidence for the dependence of normal heart protein synthesis from normal serum levels of insulin, amino acids, fatty acids and glucose. A decreased serum concentration of these substances causes an inhibition of heart muscle protein synthesis by 30--60%. Various drugs and other chemical lead to similar impairments of heat muscle protein synthesis. The resulting imbalance between synthesis and degradation of myocardial proteins with their half-times of 5--12 days gradually leads to a decrease in their myocellular concentration with a consequent impairment of myocardial function. Finally, the biochemial sequences are described which represent the important pathogenetic mechanisms in the development of heart muscle hypertrophy and in the adriamycin-induced cardiomyopathy.SummaryThe regulation of protein synthesis in heart muscle has been investigated by many authors under both normal and pathological conditions. This review summarizes the evidence for the dependence of normal heart protein synthesis from normal serum levels of insulin, amino acids, fatty acids and glucose. A decreased serum concentration of these substances causes an inhibition of heart muscle protein synthesis by 30–60%. Various drugs and other chemicals lead to similar impairments of heart muscle protein synthesis. The resulting imbalance between synthesis and degradation of myocardial proteins with their half-times of 5–12 days gradually leads to a decrease in their myocellular concentration with a consequent impairment of myocardial function. Finally, the biochemical sequences are described which represent the important pathogenetic mechanisms in the development of heart muscle hypertrophy and in the adriamycin-induced cardiomyopathy.ZusammenfassungDie Regulation der Herzmuskelproteinsynthese unter normalen und pathologischen Bedingungen wird seit langem von verschiedenen Arbeitsgruppen untersucht. In der vorliegenden Übersichtsarbeit wird die starke Abhängigkeit der Herzmuskelproteinsynthese von normalen Serumkonzentrationen an Insulin, Aminosäuren, Fettsäuren und Glukose demonstriert. Eine Verminderung dieser Substanzen unter das Niveau ihrer normalen Serumkonzentration führt zu einer Inhibierung der Herzmuskelproteinsynthese um 30–60%. Ähnliche Beeinträchtigungen werden unter der Einwirkung verschiedener Medikamente und anderer Noxen beobachtet. Bei der relativ kurzen Halbwertszeit der myokardialen Proteine von 5–12 Tagen bedingt dies ein Ungleichgewicht zwischen ihrer Synthese und Degradation mit einem konsekutiven Abfall ihrer myokardialen Konzentration. Am Beispiel der Herzmuskelhypertrophie sowie der Adriamycin-Kardiomyopathie werden abschließend die biochemischen Reaktionssequenzen beschrieben, die in der Pathogenese dieser beiden Krankheitsbilder als entscheidend angesehen werden müssen.

Quantitation of cardiac polysomal mRNA by hybridization to (3H)Poly(U)

Abstract ( 3 H)Poly(U) was hybridized to rat cardiac polysomal mRNA to determine the Poly(A) content of cardiac polysomal mRNA and to investigate whether such a hybridization assay would allow a quantitative determination of the cardiac mRNA content. The following results were obtained: 1. (a) The average Poly(A)-tract length of cardiac polysomal mRNA is 88 nucleotides, its total sequence length about 2050 nucleotides (=4.3% of the average cardiac mRNA sequence is Poly(A)). 2. (b) The rapid formation of hybrids between ( 3 H)Poly(U) and cardiac polysomal mRNA (hybrids formed in seconds to minutes) and the melting point of the hybrids (53°C) indicate that the Poly(A)-tract of cardiac mRNA is very homogeneous. 3. (c) The hybridization assay described makes it possible to quantitate cardiac mRNA in nanogram amounts allowing a quantitative determination of cardiac mRNA contents in samples of 0.5 to 2.5 mg of cardiac tissue (homogenates). This, of course, will potentially permit an application of this method to cardiac biopsies.

Quantitation of poly(A)-containing mRNA in rat cardiac biopsies

SummaryUsing a combination of conventional mRNA-extraction methods and newer hybridization techniques, a method was developed which allows the quantitation of cardiac Poly(A)-containing mRNA in cardiac biopsies. This provides a new tool to directly assess changes in cardiac gene expression in cardiac biopsies. It is proposed that the method described may be valuable in investigations dealing with various human or animal heart diseases where changes in cardiac gene expression are expected and where only biopsy material is available.ZusammenfassungDurch Kombination konventioneller mRNS-Isolierungsverfahren mit neueren mRNS-Hybridisierungstechniken wurde eine Methode entwickelt, die die Quantifizierung myokardialer, Poly(A)-enthaltender mRNS in Myokardbiopsien ermöglicht. Dadurch wird es möglich, Veränderungen der myokardialen Genexpression direkt in Myokardbiopsien zu untersuchen. Die beschriebene Methode kann daher besonders nutzvoll sein, wenn Veränderungen der myokardialen Genexpression bei menschlichen oder tierischen Herzmuskelerkrankungen erwartet werden und wenn andererseits aber nur Herzmuskelbiopsien für die Untersuchungen zur Verfügung stehen.

Tonische und phasische Aktivität des endokrinen Herzens bei kardiovaskulären Patienten und Patienten mit Leberzirrhose

Das volumensensorische und volumenregulatorische Vorhoforgan besteht aus einem afferenten Schenkel mit volumen- bzw. dehnungsempfindlichen Rezeptoren (19, 26) („to sense the fullness of the blood stream“ (34)) und einem efferenten Schenkel. Spezialisierte myoendokrine Zellen (17) konstituieren den atrialen sekretorischen Apparat; sie synthetisieren und sezernieren ein endogenes Saluretikum mit vasodilatierenden Eigenschaften, den atrialen natriuretischen Faktor (ANF) (1, 12, 24, 30, 32, 37). Neben neuronalen und anderen humoralen Effektoren (27) ist der ANF moglicherweise ein Hauptpfeiler der efferenten kardio-(zerebro-)renalen Achse (5).