Cathy J. Beinlich

Angiotensin II and left ventricular growth in newborn pig heart

The left ventricle of the neonatal pig heart is a model of rapid physiological cardiac growth that is dependent upon accelerated ribosome formation and increased RNA content. The goals of the present study were to investigate the role of angiotensin II in this rapid growth. Hearts from 3 d old control piglets or piglets that were treated with enalapril maleate, an angiotensin converting enzyme inhibitor, or DuP 753, an angiotensin II receptor antagonist, were used for measurements of left ventricular mass, RNA, DNA and protein. Hearts from enalapril-treated pigs also were used for measurements of rates of ribosome formation and total protein synthesis during perfusion as modified Langendorff preparations. Treatment of piglets with enalapril maleate resulted in decreased left ventricle/body wt ratio, RNA content, total RNA and total protein in the left ventricle. These parameters were unaffected in the right ventricle. In vitro perfusion of hearts from enalapril-treated piglets revealed decreased ribosome formation and total protein synthesis in the left ventricle. Piglets treated with DuP 753 had decreased left ventricle/body wt ratio as well as decreased RNA content, total RNA and RNA/DNA ratio in the left ventricle. These results suggest that angiotensin II may be required for rapid growth of neonatal pig hearts.

Mechanisms of rapid growth in the neonatal pig heart

During the first 2 weeks of life the left ventricular free wall of the neonatal pig heart grows rapidly. The mass of the left ventricular free wall (LVFW) increased from 2.22 +/- 0.10 g to 9.62 +/- 1.01 g while the right ventricular free wall (RVFW) increased from 2.03 +/- 0.24 g to 3.56 +/- 0.41 g from birth to 14 days of age. During the same period, the cellular volume of myocytes from the LVFW increased from 1075 microns3 to 3688 microns3 while myocytes from the RVFW increased in volume from 1511 microns3 to 2454 microns3. The number of RVFW myocytes did not change during the first 2 weeks of life, while the number of LVFW myocytes increased 28%. Myocytes from both ventricles were approximately 90% mononuclear from birth to 4-5 days of age. By 14 days, 67% of LVFW myocytes and 53% of RVFW myocytes were multinucleated. When growth of the heart was restrained by treatment of the piglet with enalapril maleate, the LVFW mass was reduced by 24% over 2 weeks compared to hearts from untreated piglets and was accounted for by a reduction in myocyte volume. Enalapril treatment did not alter the number of myocytes in either the LVFW or RVFW as compared to hearts from untreated piglets. After 14 days of enalapril treatment, the percentage of multinucleated cells was reduced in the LVFW and unchanged in the RVFW as compared to hearts from untreated piglets.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of growth in neonatal pig hearts

The pig heart grows at a maximal rate in the first 2–3 days of life due to a volume overload imposed on the heart at birth. Rates of ribosome formation and protein synthesis cannot be further accelerated duringin vitro perfusion with agents that increase cyclic AMP, that bind to α1-adrenergic receptors or that bind to angiotensin II receptors. Growth of the heartin vivo can be restrained by treatment with an angiotensin-converting enzyme inhibitor, enalapril maleate, or an angiotensin receptor antagonist, DuP 753. In the enalapril-treated heart, norepinephrine plus propranolol, but not angiotensin II, accelerated ribosome formation. Rapid growth of the left ventricle of pig heart during the first 10 days of life is due largely to eccentric hypertrophy.

Neutral-alkaline proteases and protein degradation in rat heart

Abstract The role of neutral and alkaline proteases in protein degradation in heart muscle cells was assessed by measuring rates of proteolysis in perfused rat hearts, heart muscle cells, and homogenates of hearts from control rats and rats that were treated with Compound 48 80 , a mast cell degranulator. Endogenous protein degradation in perfused hearts or in suspensions of heart muscle cells, as assessed by release of phenylalanine or disappearance of activity of S-adenosylmethionine decarboxylase in the presence of cycloheximide, was unaffected by treatment of the rat with Compound 48 80 . The rate of phenylalanine release in heart muscle cells was similar to the rate that was observed in perfused hearts. Homogenates of control hearts that were incubated in the presence of Triton X-100 released phenylalanine over a pH range from 5 to 8 at rates that were equal to or greater than the rate of protein degradation that was observed in perfused hearts. At pH 5, only 30% of phenylalanine release was assayable in the absence of detergent. Homogenates of hearts from 48 80 - treated rats and heart muscle cells released phenylalanine at pH 5 at the same rate as control homogenates, but at pH 7.8 the rate was only 10% of the value of control homogenates. These studies indicated that a large fraction of the alkaline proteolytic activity was in non-muscle cells and that this activity contributed little to overall protein degradation. These findings emphasize the importance of identifying the cellular localization of proteases before attempting to assess their role in myocardial proteolysis.

Contributions of increased efficiency and capacity of protein synthesis to rapid cardiac growth

Rapid cardiac growth depends upon faster synthesis than degradation of protein. The rate of protein synthesis is determined by the efficiency with which the existing components of the ribosome cycle make protein and by the quantity of the components that are present. The tissue content of RNA is taken as an index of the capacity of synthesis and efficiency is expressed as the amount of protein formed per amount of RNA over a certain time period. The efficiency of synthesis is regulated by hormones, including insulin, agents that increase cAMP, α-adrenergic agonists, endothelin I and angiotensin II. In addition, provision of non-carbohydrate substrates and mechanical factors such as stretch and contraction increase efficiency. Impaired energy availability as occurs in anoxic or ischemic muscle decreases efficiency. Increased phosphorylation of ribosomal protein, S6, or of the peptide chain initiation factor, elF-4E, have been suggested as mechanisms to regulate efficiency of mRNA translation. Increased efficiency of synthesis accounts for cardiac growth in the first few days following aortic banding, pulmonary artery constriction and thyroxine administration. Decreased efficiency accounts for cardiac atrophy in heterotopic transplanted hearts during the first 3 days following transplantation. The capacity of synthesis is increased by insulin, thyroid hormone, activators of protein kinase C, agents that increase cAMP, and endothelin-1. Stretch of the ventricular wall and contraction of cultured neonatal myocytes accelerates ribosome formation. An increased rate of ribosomal DNA transcription accounts for accelerated ribosome formation and depends on increased activity of a transcription factor, upstream binding factor (UBF). The activity of UBF is increased either by increased rates of synthesis or by phosphorylation of the protein. Increased capacity of synthesis is a major contributor to rapid cardiac growth in the newborn heart and after several days of pressure overload.

Myocardial metabolism of pantothenic acid in chronically diabetic rats

Transport and metabolism of [3H]pantothenic acid ([3H]Pa) was investigated in hearts from control and streptozotocin-induced diabetic rats. In isolated perfused hearts from control animals, the transport of [3H]Pa was linear over 3 h of perfusion when 11 mM glucose was the only exogenous substrate. The in vitro transport of [3H]Pa by hearts from 48-h diabetic rats was reduced by 65% compared to controls and was linear over 2 h of perfusion with no further accumulation of Pa during the third hour. The defect in transport observed in vitro could be corrected by in vivo treatment with 4 U Lente insulin/day for 2 days. In vitro addition of insulin in the presence of 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate had no effect on [3H]Pa transport in hearts from 48-h diabetic rats during 3 h of perfusion. Accumulation of [3H]Pa was not inhibited by inclusion of 0.7 mM amino acids, 1 mM carnitine, 50 microM mersalic acid or 1 mM panthenol, pantoyllactone or pantoyltaurine. Uptake was inhibited by 1 mM nonanoic, octanoic or heptanoic acid, 0.1 mM biotin or 0.25 mM probenecid, suggesting a requirement for the terminal carboxyl group for transport. Transport of pantothenic acid was reduced in hearts from diabetic rats within 24 h of injection of streptozotocin. In vitro accumulation of [3H]Pa decreased to 10% of control 1 week after streptozotocin injection and then remained at 30% of the control value over 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of growth in the neonatal pig heart

The newborn heart is an excellent model in which to study cardiac growth because the neonatal period is a normal situation in which the left ventricle (LV) grows rapidly and the right ventricle grows slowly. Accelerated LV growth is in response to mechanical, neural, and endocrine changes at birth. Faster growth of the LV is accounted for by greater capacity for protein synthesis, as evidenced by greater RNA content. At 18 h of life, ribosomes are formed in preference to total heart protein, but at 48 h of life, faster rates of both ribosome formation and total protein synthesis are observed. In the LV of hearts from 2-day-old pigs, these rates are insensitive to the addition of glucagon, 1-methyl-3-isobutylxanthine, or a combination of norepinephrine and propranolol. These observations could result because of maximal growth stimulation already present in the LV of the newborn heart. To restrain LV growth in the neonatal period, we treated pigs with enalapril maleate, an angiotensin II-converting enzyme inhibitor. Enalapril blocked growth of the LV as well as the increase in RNA content. When hearts from enalapril-treated pigs were perfused in vitro, rates of protein synthesis and ribosome formation in the LV were lower. These studies suggest that angiotensin II is an important factor accounting for rapid growth of the neonatal heart in response to pressure overload at birth.

Metabolism of pantothenic acid in hearts of diabetic rats

The metabolism of pantothenic acid (Pa) by cardiac muscle was studied in normal and diabetic rats. Tissue levels of Coenzyme A (CoA) are elevated in the heart during early (6 to 12 h) diabetes, remains at a high level for several days, and then returns to normal or below normal levels. The increase in total tissue CoA mainly occurs in myocytes as indicated by isolation of cardiac myocytes from control and diabetic animals and measuring their content of CoA. The CoA concentration increased from 37 to 93 microM in the cytosolic compartment and from 2.0 to 2.6 mM in the mitochondrial matrix. These effects of diabetes were reversed by insulin treatment. CoA synthesis in hearts removed from control rats and perfused in vitro was stimulated by including in the perfusate Pa, cysteine and dithiothreitol, but no exogenous energy substrate. This stimulated in vitro rate of CoA synthesis was reduced in hearts removed from diabetic animals, and the reduction increased with duration of diabetes. The reduced rate in diabetic hearts resulted from both a decreased rate of Pa phosphorylation and decreased Pa transport. Transport of Pa into myocytes was decreased by as much as 80% in hearts from diabetic animals. The low transport rate was due to a decrease in Vmax with no apparent change in Km. Treatment of the isolated heart with insulin did not correct the diabetic-induced reduction in Pa transport. The transport rate in normal and diabetic hearts was not influenced by the type of energy substrate provided to the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of bradykinin in the antihypertrophic effects of enalapril in the newborn pig heart

Rapid growth of the left ventricle of the newborn pig heart can be restrained by treating piglets with the angiotensin converting enzyme inhibitor, enalapril maleate. This reduced rate of growth is reflected in vitro by reduced rates of ribosome formation and protein synthesis, and may be due to decreased availability of angiotensin II (All), a potentially hypertrophic agent; decreased numbers of All receptors; increased availability of bradykinin, a potentially antihypertrophic agent; or reduced hemodynamic load on the left ventricle. Because enalapril decreases degradation of bradykinin, the role of bradykinin as an inhibitor of cardiac growth in the newborn heart was investigated. Addition of 1 × 10−5 M bradykinin and 1 × 10−6 Menalapril to the perfusate of isolated hearts from 2 day old piglets did not significantly alter heart rate, contents of ATP or creatine phosphate or rates of ribosome formation or protein synthesis during 1 h of perfusion. Similarly, exposure of myocytes isolated from the left ventricular free wall of piglets to 5 × 10−6 M bradykinin for 72 h did not alter the rate of [3H]-phenylalanine incorporation into total protein. The reduced rate of left ventricular growth in vivo caused by enalapril administration was not reversed by simultaneous treatment with the specific bradykinin receptor antagonist, HOE 140. HOE 140 alone did not alter ventricular growth as compared to hearts from untreated piglets. In summary, these results demonstrate that the reduced rate of left ventricular growth in vivo and the reduced rate of ribosome formation and protein synthesis in the left ventricle in vitro after enalapril treatment of piglets is not the result of an inhibitory effect of bradykinin on cardiac growth.

Neutral-alkaline proteolytic activity in rat cardiac muscle cells

Homogenates that were prepared from isolated heart muscle cells were found to have reduced ability to degrade endogenous proteins to free amino acids at alkaline pH in comparison to homogenates of whole hearts. The decrease in alkaline proteolytic activity was the result of loss from the particulate fraction. The heart muscle cell preparations retained considerable proteolytic activity at neutral-alkaline pH as indicated by the hydrolysis of [3H]acetylcasein and [14C]protein from rat heart to acid soluble products. In both cases, the decrease in activity was associated with the particulate fraction and appeared to be the result of the absence of mast cells. The extractable activity of control hearts and heart muscle cells eluted from DEAE-cellulose columns as two peaks of caseinolytic activity. The first peak eluted with 0.07 to 0.12 m NaCl and included a thiol protease. The second peak eluted with 0.25 m NaCl and was seen only in the presence of 5 mm CaCl2. The specific activity of the Ca2+-dependent protease was reduced 50% in muscle cell preparations as compared to control heart.