Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms.

The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.

Hypertrophied rat heart. New Na+, K+-ATPase-ouabain interactions in sarcolemma vesicles.

The interactions between ouabain and sarcolemmal-bound Na+, K+-ATPase activity have been studied in rat left ventricles 70-130% hypertrophied by aortic constriction. Isolated sarcolemmal vesicles were 75-fold enriched in Na+, K+-ATPase and consisted in both impermeable inside-out vesicles and leaky material. The enzymatic activity was found to be almost completely inhibited at 10(-6) M ouabain instead of 2 X 10(-4) M in the normal left ventricle. In contrast with normal heart in which a Ca2+-free perfusion revealed a Na+, K+-ATPase activity highly sensitive to ouabain, in hypertrophied heart, a pretreatment plus or minus Ca2+ did not affect the high sensitivity form (half maximum inhibition with 0.8-2.2 X 10(-9) M ouabain). This enzyme form differed from the usual Na+, K+-ATPase activities by the absence of any detectable ouabain/K+ antagonism. The levels of 3H-ouabain binding, however, did not correlate with the levels of Na+, K+-ATPase inhibition. These results suggested that the Na+, K+-ATPase-ouabain interactions were altered in hypertrophied rat heart.

Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart.

Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.

The incorporation of radioactive lysine or tyrosine into cardiac and skeletal myofibrillar and non-myofibrillar contractile proteins.

The labelled amino acids incorporation into cardiac and skeletal contractile proteins has been compared after a single injection of 3H lysine, repeated injections of 3H lysine during 1 or 6 hours or after a continuous infusion of both 3H-lysine and 14C-tyrosine. The myofibrillar incorporation was higher in the heart than in the skeletal muscle. Myosin heavy chains and actin have been prepared using gel filtration. The incorporation was again higher in the heart for both these proteins but the labelling of actin in both the muscles reaches rapidly a plateau in contrast with myosin, suggesting that these two proteins possess a different precursor pool. Myosin heavy chains prepared from the supernatant obtained after a relaxing treatment were more labelled than those extracted from myofibrils. These heavy chains from the supernatant were presumably newly synthetized and not yet incorporated into myofibrils. They also were more labelled in the heart than in the skeletal muscle which means that the myosin synthesis itself was different and not the process of assembly of the myofibrils.

Absence of phosphates from the myosin heavy chains of striated muscles.

This work aimed to determine whether the heavy chains of myosin from different striated muscle were phosphorylated. Myosin and its heavy chains were prepared from cardiac and skeletal muscles of rats injected in vivo with radioactive phosphates. The results for radioactive phosphate localization indicate the absence of phosphate from pure heavy chains and from any of their purified fragments, whatever the striated muscle used. In addition, phosphates are present in the myosin phosphorylated light chain and in a contaminating protein closely associated to the myosin heavy chain.

The relationship of a decline in myofibrillar ATP-ase activity to the development of severe left ventricular hypertrophy in the rat.

Abdominal aortic coarctation in the rat led to a modest degree of cardiac hypertrophy associated with normal ATPase activity of myofibrills. The addition of aortic incompetence was accompanied by the development of severe hypertrophy (approx. 75 %). During the course of this latter increase in heart weight, the Ca++ ATPase of myofibrils progressively declined to a level 36% below controls. This occurred at a time when protein synthesis was stimulated as evidenced by increased incorporation of H3 lysine into myofibrils. Results are compatible with the hypothesis that the abnormal ATPase activity reflects the synthesis of an abnormal myosin molecule.

Cardiac myosin: preparation, ATPase in chronic heart hypertrophy.

A low myofibrillar ATPase seems to be established definitely in several experimental models of chronic heart hypertrophy as well as in humans, but the biochemical pathogenesis of this defect is still unclear. Three different preparations of myosin were studied. Their purity was estimated by measuring MgATPase or by polyacrylamide gel electrophoresis. The first preparation was highly contaminated by actin and tropomyosin; the second was rather pure, and the third (chromatography on DEAE-Sephadex) was pure but slightly denaturated. Heart myosin CaATPase (ionic strength 0.6 or 0.06) was decreased in chronic aortic insufficiency in the rabbits (CAI) when all three preparations were tested. Two (molecular weight 18,000 and 26,000), sometimes three light subunits were found in heart myosin. Their charge and molecular weight are normal in CAI. The third subunit (molecular weight 15,500) was found in control as well as in CAI. Search for an inhibitor was unsuccessful since the two myosin ATPases are additive. The nucleoprotein peak separated from myosin during chromatography was identical in control and CAI. Therefore, myosin seems to be abnormal in CAI.