Comparison of the electromechanical responsiveness of alpha-1-adrenoceptor stimulation in ventricles of normal and cardiomyopathic hamsters

Alterations in alpha(1)-adrenoceptor (alpha(1)AR) density and related signal transduction proteins were reported in cardiomyopathic hearts in the failing stage. The electromechanical modification of alpha(1)-adrenergic stimulation in the failing heart is unclear. The present study compares the alpha(1)AR-stimulated electromechanical response in failing ventricles of genetically cardiomyopathic BIO 14.6 hamsters (280-320 days old) with that in age-matched normal Syrian hamsters. The action potential was recorded with a conventional microelectrode technique, and twitch force was measured with a transducer. In the presence of propranolol, phenylephrine increased the contraction and prolonged the action potential duration (APD) to similar values in ventricles of both strains, despite a prolonged basal APD in cardiomyopathic ventricles. The positive inotropism stimulated by phenylephrine was inhibited by staurosporine, and was potentiated by 4 beta-phorbol-12,13-dibutyrate (PDBu) in both strains. The maximum positive inotropic effect of phenylephrine in PDBu-treated ventricles of normal hamsters was significantly greater than that in BIO 14.6 hamsters. The effects of phenylephrine on the ventricular force-frequency relationship and on the mechanical restitution in both normal and BIO 14.6 strain hamsters were examined. The uniform negative force-frequency relationship and the altered mechanical restitution reveal a defect of intracellular Ca(2+) handling in cardiomyopathic BIO 14.6 hamsters. alpha(1)-Adrenergic modulation cannot convert the defective properties in the model of the failing heart. Nevertheless, phenylephrine decreased post-rest potentiation in short rest periods, and enhanced post-rest decay after longer resting periods. The results indicate that alpha(1)-adrenergic action enhances a gradual loss of Ca(2+) from the sarcoplasmic reticulum, although its action in prolonging the APD can indirectly increase the influx of Ca(2+).     

Differential tissue expression and developmental regulation of guanine nucleotide binding regulatory proteins and their messenger RNAs in rat heart

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.     

Increase in the relative abundance of preproenkephalin A messenger RNA in the ventricles of cardiomyopathic hamsters

Preproenkephalin A messenger RNA was detected in hamster heart by Northern blot analysis using a human preproenkephalin A cDNA probe. Ventricular levels of this messenger were one order of magnitude lower than atrial levels, which were equivalent to brain levels. Furthermore, in the heart of cardiomyopathic hamsters, an animal model of cardiac hypertrophy and congestive heart failure, the relative abundance of the preproenkephalin A messenger RNA was found to increase three- to four-fold in ventricles while no change was seen in atria. These results support the hypothesis that the heart has the potential for locally synthesizing enkephalins and provide evidence that alterations in preproenkephalin A messenger RNA levels are associated with the development of cardiac hypertrophy and failure.     

Identification of a novel aldose reductase-like gene upregulated in the failing heart of cardiomyopathic hamster

Cardiomyopathy (CM) is degenerative disease of myocardium which leads to severe cardiac failure. Although many causative genes for CM have been identified, molecular pathogenesis of CM is not fully understood. In this study, we searched for a novel pathway recruited in the development of CM by using BIO14.6 hamster as an animal model for human CM. We screened upregulated genes in the left ventricle by differential display technique and searched for a gene which had never been linked to CM. We identified a novel gene overexpressed in BIO14.6 hamster ventricles, which was considered to be a new member of aldo-keto reductase (AKR) superfamily. The cloned cDNA encoded a 316 amino acid polypeptide with calculated molecular mass of 35,804, which showed high amino acid sequence similarities to aldose reductase and its relative: 69.6% to AKR1B1 (human aldose reductase), 68.4% to AKR1B3 (mouse aldose reductase), and 85.8% to AKR1B7 (mouse vas deferens protein). The upregulation of this aldose reductase-like gene in BIO14.6 hamster ventricles (6.3 ± 0.8-fold) seemed to be influenced by the overexpression of activator protein-1 present there. With the fact that AKR1B1, AKR1B3, and AKR1B7 have synthetic activities of prostaglandin F2α, the aldose reductase-like protein could cause cardiac hypertrophy through production of prostaglandin F2α whose precursor and receptor were abundant in BIO14.6 hamster ventricles. Aldose reductase and its related proteins would give a new clue to dissect the pathogenesis of CM including oxidative stress and cardiac hypertrophy, and to develop a new drug for the treatment of CM.     

Atrial natriuretic factor in taurine-treated normal and cardiomyopathic hamsters

Diuretic and natriuretic activities of atrial extracts from BIO 14.6 (cardiomyopathic) and F1B (normal) hamsters at 180 days of age were measured by rat bioassay. Both activities were lower in BIO 14.6 extracts. Because of the reported protective action of taurine in the cardiomyopathic hamster, we tested the effect of 0.1 M taurine drinking upon the activity of atrial extracts. Urine flow and Na+ excretion were increased in both BIO 14.6 and F1B; however, comparatively larger increases in BIO 14.6 taurine drinkers abolished strain differences that were observed in water drinkers. Taurine drinking BIO 14.6 hamsters exhibited an increased plasma sodium concentration. Drinking of 0.6% NaCl also produced an elevated plasma sodium concentration in BIO 14.6. Extracts from hamsters with increased salt intake had diuretic and natriuretic activities that were not different from those of water drinkers. These findings confirm that ANF activity is deficient in BIO 14.6 hamsters, and this suggests a role for taurine in its production, release, and/or activation.     

Light and electron microscopic localization of atrial natriuretic peptide in the heart of spontaneously hypertensive rat

Atrial natriuretic peptide (ANP) is a newly discovered peptide hormone present mainly in the atria. We investigated the occurrence and distribution of ANP immunoreactivity in the myocardiocytes of the ventricles of spontaneously hypertensive rats by use of immunocytochemistry at both light and electron microscopic level. ANP immunoreactivity was found in the specific granules in the cytoplasm of the cardiocytes in the subendocardium and the myocardium of the ventricles, as well as in the atria. The specific granules found in the ventricles of hypertensive rats were similar in size, shape, and ANP immunoreactive content to those in the atria. The abundance of ANP immunoreactivity in the left ventricle is greater than that in the right, and appears to increase with increasing severity of hypertension. Conversely, the overall content of ANP in the atria of hypertensive rats was decreased when compared with that in age-matched normotensive rats. The present findings indicate that ventricles may become a major source for ANP synthesis and release during hypertension, and may play important roles in cardiac endocrine pathology and cardiac hypertrophy.     

Cardiac-specific Deletion of LKB1 Leads to Hypertrophy and Dysfunction

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using α-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.     

Cloning and expression of the atrial natriuretic factor gene

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.     

Atriopeptin release in conditions of altered salt and water balance in the rat

Manipulations of salt and water intake influenced the atriopeptin content in the atria and plasma of rats. Plasma levels of atriopeptin varied in proportion with dietary salt intake. In contrast, cardiac levels of atriopeptin varied inversely with the amount of salt in the diet. Acute stimulation of atriopeptin release can be produced by treatments which elevate atrial pressure, including atrial stretch, volume overloading, water immersion, and vasoconstrictor agents. Vasopressin-stimulated atriopeptin release preferentially depleted right atrial stores. In spite of the initial differences in cardiac stores of atriopeptin in the rats on different diets, there were no major differences in the amount of atriopeptin released in response to vasopressin stimulation. These data suggest that there is a functional excess of cardiac atriopeptin stores. We also examined the atrial and plasma atriopeptin content in the Dahl salt-sensitive and resistant rats to determine whether the development of hypertension in the Dahl sensitive rats is associated with abnormalities in basal or stimulated levels of atriopeptin. The effects of dietary salt intake on basal and stimulated atriopeptin levels in both the Dahl sensitive and resistant rats were similar to those observed in normal rats, suggesting that abnormalities in atriopeptin content do not contribute to the etiology of hypertension in the Dahl salt-sensitive rat.     

Elevated plasma vasopressin in cardiomyopathic hamsters

Hamsters of the BIO 14.6 strain characteristically develop cardiomyopathy as they age, and hamsters of this strain have overt signs of heart failure by 11 months of age. Plasma levels of the posterior pituitary hormone arginine-vasopressin (AVP) were found to be elevated (approximately 2-fold) in 11 month old BIO 14.6 hamsters, compared to age-matched hamsters of a control strain. AVP appeared inappropriately elevated in these animals, since they were neither hyperosmotic nor markedly hypotensive. The elevated levels of AVP observed in these animals appears to contribute to vasomotor tone, since intravenous adminstration of a specific antagonist of the vasoconstrictor action of AVP [d(CH₂)₅Ome(TYR)AVP] elicited a fall in arterial pressure (9±2 mm Hg, n=6, p<0.05). The AVP antagonist had no effect on arterial pressure in hamsters of a control strain, and vehicle administration had no effect on arterial pressure in either strain. These data indicate that inappropriately elevated levels of AVP contribute to the cardiovascular state of myopathic hamsters. Since elevated plasma AVP has been noted in human congestive heart failure, these results suggest that AVP may contribute to the cardiovascular status during congestive heart failure.     

Quantitative studies on the ultrastructural differentiation and growth of mammalian cardiac muscle cells. The atria and ventricles of the cat

Ultrastructural differentiation of cardiac muscle cells in the bilateral atria and ventricles of the cat at 1, 16, 25 and 40 days and 6 months after birth was studied by morphometry on electron micrographs. At the newborn stage, no T-tubule was found in the ventricular muscle cells, but specific granules were already noted in the atrial myocytes. The cell diameter of the ventricular myocardium was greater than that of the atrium at this stage. The T-tubule was first recognized in the ventricular muscle cells at day 16, at which stage the area occupied by the mitochondria and glycogen in the atrial muscle cells was definitely found to differ from that in the ventricular muscle cells. Thereafter, the differences in the ultrastructure between the atria and ventricles became more remarkable, particularly in the cell diameter and in the mitochondrial area. The cat cardiac muscle cells are characterized by numerous lipid droplets within the cytoplasm in contrast to those of the rat and the guinea pig.     

Tissue-specific regulation of GTP-binding protein and muscarinic acetylcholine receptor levels during cardiac development

A quantitative immunoblot assay was developed by using affinity-purified monospecific antibodies to quantitate levels of guanine nucleotide binding regulatory protein (G-protein) subunits in atria and ventricles during embryonic chicken cardiac development. The muscarinic acetylcholine receptor (mAChR) number was measured with [3H]quinuclidinyl benzilate. On day 10 of embryonic development (day 10E) there was no difference between the atrial and ventricular membrane concentrations of beta-subunit, G0 alpha subunit, or mAChR. The level of Gi alpha was found to be 44% greater in atria than in ventricles on day 10E. The atrial membrane concentration of beta-subunit increased 80% between day 13E and 15E, G0 alpha increased 46% between day 10E and 15E, mAChR increased 61% between day 10E and 12E, and Gi alpha decreased 34% between day 10E and 13E. The atrial levels of beta-subunit, G0 alpha, Gi alpha, and mAChR did not change further through day 20E. The ventricular membrane concentration of these proteins did not change between day 10E and 20E, except for that of G0 alpha, which increased 47% between day 15E and 20E. The atrial specific increase in beta-subunit correlated with a loss of GTP inhibition of basal adenylate cyclase activity. The difference in Gi alpha levels between atria and ventricles on day 10E correlated with a difference in carbachol sensitivity of atrial and ventricular basal adenylate cyclase activity. Thus, the levels of several components of the cholinergic neuroeffector pathway are regulated in a tissue-specific manner at a time that coincides with the onset of functional parasympathetic innervation of the embryonic chicken heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Differential regulation of SR calcium transporters by thyroid hormone in rat atria and ventricles

Thyroid hormone exerts positive inotropic effects on the heart mediated in part by its regulation of calcium transporter proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2), phospholamban (PLB), and Na(+)/Ca(2+) exchanger (NCX). To further understand the potential cardiac chamber-specific effects of thyroid hormone action, we compared the triiodo-L-thyronine (T(3)) responsiveness of calcium transporter proteins in atrial versus ventricular tissues. Rats were rendered hypothyroid by ingestion of propylthiouracil, and a subgroup of animals was treated with T(3) for 7 days (7 microg/day by constant infusion). Atrial and left ventricular (LV) tissue homogenates were analyzed for expression of SERCA2, PLB, and NCX proteins by Western blot analysis. SERCA2 protein significantly decreased by 50% in hypothyroid LV and was normalized by T(3) treatment. In contrast, SERCA2 protein in atria was unaltered in the hypothyroid state. PLB protein expression significantly increased by 1.6- and 5-fold in the hypothyroid LV and atria, respectively, and returned to euthyroid levels with T(3) treatment. Expression of NCX protein showed a greater response to T(3) treatment in atria tissue than in ventricular tissue. Sarcoplasmic reticulum calcium cycling is determined in part by the ratio of SERCA2 to PLB. This ratio was sixfold higher in the atria compared with LV, suggesting that PLB may play a minor role in the regulation of SERCA2 function in normal atria. We conclude that calcium transporter proteins are responsive to thyroid hormone in a chamber-specific manner, with atria showing a greater change in protein content in response to T(3). The differential effect on atria may account for the occurrence of atrial rather than ventricular arrhythmias in response to even mild degrees of thyrotoxicosis.     

The Role of Guanine Nucleotide Binding Proteins in Hamsters with Myocardial Hypertrophy

Abstract

To clarify the role of the guanine nucleotide binding proteins (G-proteins) in the pathogenesis of myocardial hypertrophy, we investigated the alterations in myocardial G proteins in 20-week-old F1b hamsters with pressure overload induced for 3 days (3-day AS), 7 days (7-day AS) and 14 days (14-day AS) by the stenosis of the abdominal aorta, and in 4- and 20 week-old BIO 14.6 Syrian hamsters (4-wk and 20-wk BIO) with genetic myocardial hypertrophy. The hearts of 7-day AS, 14-day AS and 20-wk BIO with left ventricular hypertrophy exhibited a decrease in the mRNA levels detected by Northem blot analysis and protein levels of G protein detected by Western blot analysis as compared with sham-operated and age-matched F1b hearts. The function of Gs or Gi showed a concomitant reduction in both models of myocardial hypertrophy. The hearts of 3-day AS and 4-wk BIO without myocardial hypertrophy showed no changes in G proteins as compared with sham-operated and age-matched F1b hearts. These results suggest that a decrease in G proteins is not involved in the pathogenesis of myocardial hypertrophy, but that myocardial hypertrophy reduced the G proteins.     

Catecholamine, dopamine-beta-hydroxylase and atrial natriuretic factor content in separate heart chambers of cardiomyopathic hamsters

Since previous investigations have suggested a relationship between atrial natriuretic factor (ANF) and dopamine-beta-hydroxylation, cardiomyopathic hamsters were studied for atrial and ventricular catecholamine (CA) and dopamine-beta-hydroxylase (D beta H) content as correlates to a parallel finding of markedly decreased atrial but increased ventricular ANF concentrations in these animals. It was noted that, with progressive cardiomyopathy, the reduced tissue norepinephrine (NE) content paralleled the declining D beta H activity in the atria. In the ventricles, however, the progressively-decreasing NE content was associated with an increase of D beta H. These data indicate that the NE depletion is mediated by different mechanisms in the ventricles and atria. They do not support a simple relationship between NE depletion and tissue D beta H activity or between the latter and tissue ANF concentrations.