Subcellular distribution of myocardial 5'-nucleotidase

The controversial subject of the subcellular location of myocardial adenosine production was studied employing density gradient fractionation of heart muscle combined with a novel method for analyzing distribution profiles based on multiple regression (correlation) analysis. Bungarotoxin binding, N-acetyl-beta-D-glucosaminidase, cytochrome c oxidase, NADPH-dependent cytochrome c reductase and lactate dehydrogenase were used as markers for the plasma membrane, lysosomes, mitochondria, sarcoplasmic reticulum and cytosol, respectively. The normalized distribution frequencies (fraction of total) of 5'-nucleotidase in mitochondria, lysosomes, plasma membranes, sarcoplasmic reticulum and cytosol in the 50 x g supernatant of total homogenate of heart muscle were found to be 0, 0.25, 0.44, 0.08 and 0.23, respectively. To increase the resolution power of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied, in which the normalized distribution of 5'-nucleotidase in the homogenate was 0, 0.16 and 0.84 in mitochondria, plasma membranes and lysosomes, respectively. This mainly lysosomal 5'-nucleotidase activity was 61% inhibited by the alpha,beta-methylene analog of ADP, indicating that although the latter has been considered specific to the plasma membrane enzyme, it also inhibits the lysosomal enzyme. The intercellular distribution of 5'-nucleotidase was not studied, but the lack of this enzyme in the mitochondria indicate that the adenosine production observed during mitochondrial AMP production, e.g. during acetate oxidation in intact heart muscle, must involve AMP transport out from the mitochondria.     

Cardiac contractile proteins in hypertrophied and failing guinea pig heart.

OBJECTIVE: The aim was to study changes in contractile proteins which accompany marked hypertrophy and heart failure in mammalian hearts initially containing predominantly V3 isomyosin. METHODS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were measured in normal guinea pig hearts, in hearts which were hypertrophied as a result of progressive left ventricular systolic overload following ascending aortic banding, and in hypertrophied hearts from animals which showed signs of overt congestive heart failure. Male guinea pigs weighing 225-275 g at the time of aortic banding were used for the studies. RESULTS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were correlated with body weight, left and right ventricular weight, and left ventricular peak systolic pressure during aortic occlusion. Left ventricular myosin ATPase activity and right ventricular actomyosin ATPase activity were markedly depressed in hypertrophied ventricles compared to control ventricles. Cardiac myofibrillar ATPase activity was lower in hypertrophied failing hearts than in control hearts over a wide range of calcium concentrations. In control animals and in those without heart failure, there was a nearly identical inverse relationship between left ventricular mass up to 1600 mg and myosin ATPase activity. Hypertrophied failing hearts were larger but showed little further reduction in cardiac myosin ATPase activity. Representative gel scans of non-dissociating pyrophosphate gels of left ventricular myosin from an 8 d postoperative aortic constricted animal and from its age and weight matched control showed predominantly V3 isomyosin with small amounts of V1 isoenzyme. However, preparations taken from guinea pigs 16 d after aortic constriction showed only the V3 isoform, whereas the V1 isoform was still apparent in control. Hypertrophied failing left ventricles developed less pressure per unit mass during brief aortic occlusion than non-failing left ventricles with comparable myosin ATPase activities. CONCLUSIONS: These observations raise important questions as to the distribution of myosin isoforms in the normal adult guinea pig, and the possibility that myosin ATPase activity might be altered by post-translational modification. Although cardiac myosin ATPase activity correlates with left ventricular performance, it cannot fully explain the depressed performance of failing hearts in this model. Additional immunological studies of cardiac contractile proteins are required as well as studies designed to explore the implications of altered myosin ATPase activity for both contractile function and overall cellular homeostasis.     

Metabolism of cardiac structural proteins in normal and hypertrophied rabbit heart

In an attempt to elucidate the characteristics of the metabolic response of the hypertrophying heart, the rates of amino acid incorporation into cardiac structural proteins were studied in female rabbit hearts in vivo by use of a pulse-label isotope (3H-lysine) method. Hypertrophy of the left ventricle was produced by applying mild aortic constriction. Structural proteins were separated from the heart excised 24 h after the pulse, and the rate of 3H incorporation was calculated as cpm. 3H/mg lysine of the protein. In the control (without aortic constriction), left ventricular structural proteins exhibited the following relative incorporation rate (the rate in actin being taken as 1.0): native tropomyosin, 2.79 +/- 0.38 (mean +/- SE, n = 5); 10S-actinin and alpha-actinin complex, 2.52 +/- 0.29; heavy chain of myosin, 2.09 +/- 0.25; light chain of myosin, 1.82 +/- 0.28; soluble protein, 1.79 +/- 0.30; and actin, 1.0. In the rabbits with aortic stenosis the rates of amino acid incorporation showed a striking increase up to 7 days after surgery, and declined to the control level by 1 month. Among the protein fractions, native tropomyosin and the light chain of myosin showed a much higher rate of isotopic incorporation during the earlier phase of the response. These results seem to indicate the presence of a unique pattern of metabolic response in cardiac muscle confronted with a workload.     

Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart.

In an attempt to determine whether a subcellular compartmentation of creatine kinase exists and if so, whether there is a different distribution of the 3 isoenzymes of CK in the cell, studies were carried out with the guinea pig heart which had been subfractionated by either isopycnic density gradient centrifugation or differential pelleting. Isoenzyme analysis of CK in the isolated subcellular fractions by electrophoresis on agarose gels, revealed that the MM isoenzyme occurred in the cytosol, myofibrils, and the microsomes while the MB isoenzyme (which is cardio-specific) was only found in the cytosol. Trace amounts of the BB isoenzyme were detected in the cytosol. Considerable CK activity was associated with the mitochondria, this did not represent the MM, the MB, or the BB isoenzymes but was a distinct and additional mitochondrial-specific form of CK. PH optima and kinetic studies were carried out to characterise and distinguish the mitochondrial isoenzyme from other CK isoenzyme activity. The evidence for a differential compartmentation of MM, MB, BB, and mitochondrial CK is discussed in relation to their possible cellular roles.     

Subcellular remodelling may induce cardiac dysfunction in congestive heart failure

It is commonly held that cardiac remodelling, represented by changes in muscle mass, size, and shape of the heart, explains the progression of congestive heart failure (CHF). However, this concept does not provide any clear information regarding the development of cardiac dysfunction in CHF. Extensive research has revealed that various subcellular organelles such as the extracellular matrix, sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, and nucleus undergo varying degrees of changes in their biochemical composition and molecular structure in CHF. This subcellular remodelling occurs due to alterations in cardiac gene expression as well as activation of different proteases and phospholipases in the failing hearts. Several mechanisms including increased ventricular wall stress, prolonged activation of the renin-angiotensin and sympathetic systems, and oxidative stress have been suggested to account for subcellular remodelling in CHF. Furthermore, subcellular remodelling is associated with changes in cardiomyocyte structure, cation homeostasis as well as functional activities of cation channels and transporters, receptor-mediated signal transduction, Ca(2+)-cycling proteins, contractile and regulatory proteins, and energy production during the development of heart failure. The existing evidence supports the view that subcellular remodelling may result in cardiac dysfunction and thus play a critical role in the transition of cardiac hypertrophy to heart failure.     

Clinical utility of gated cardiac blood pool imaging in congestive left heart failure

Our experience with gated cardiac blood pool imaging in the evaluation of congestive left-sided heart failure was reviewed in 82 patients. Ventricular contraction patterns, right and left ventricular size, and regional wall motion were evaluated from technetium-99m-albumin gated blood pool scans obtained in anterior and left anterior oblique projections. Patterns of ventricular function shown by scan were classified as follows: normal right and left ventricular size and contraction, normal left ventricular size with right ventricular enlargement, left ventricular volume overload, diffuse left ventricular hypokinesis, regional left ventricular asynergy, left ventricular aneurysm and hypertrophic cardiomyopathy. In 34 of 36 patients who underwent cardiac catheterization, the pattern of left ventricular dysfunction revealed by scan agreed with the findings on left ventriculography. Left ventricular end-diastolic diameters were significantly (p < 0.001) increased in patients with heart failure due to previous myocardial infarction, congestive cardiomyopathy, left ventricular volume overload and left ventricular pressure overload. Right ventricular diameters were increased predominantly among patients with congestive cardiomyopathy and mitral stenosis. Clinically, gated cardiac imaging was useful for (1) diagnostic screening prior to cardiac catheterization; (2) determination of the potential for improvement with surgical operation; and (3) prognostication from the severity of left ventricular dysfunction.     

Transmural distribution of extracellular purines in isolated guinea pig heart.

The purine adenosine appears to be involved in regulation of coronary vascular tone. Little is known concerning the levels and distribution of adenosine and related purines in the extracellular fluid of the heart. We have measured epicardial and endocardial levels of adenosine, inosine, hypoxanthine, AMP, and IMP in isolated constant flow perfused guinea pig hearts by using a recently developed technique with porous nylon sampling discs. Venous effluent purine levels were also measured. Concentrations of all purines measured, excluding IMP, were significantly higher in endocardial fluid samples than in epicardial fluid samples (P less than 0.05). Conversely, IMP levels were significantly lower in endocardial than in epicardial samples. The magnitude of the endocardial/epicardial ratios for adenosine, inosine, hypoxanthine, AMP, and IMP were approximately 12:1, 4:1, 5:1, 4:1, and 1:2, respectively. To assess cellular damage, lactate dehydrogenase activity was measured in all fluid samples and was not significantly different in endocardial and epicardial fluid. These data support the existence of significant transmural gradients for extracellular purine levels in crystalloid perfused guinea pig hearts. Transmural differences in vasoactive adenosine levels may be partially due to the greater endocardial oxygen consumption and metabolism and may be involved in maintaining relatively high subendocardial blood flows in the face of high intramyocardial pressures.     

Effect of pressure overload on cardiac Ca2+ antagonist binding sites of guinea pig. Comparison with the adaptational response of the hypertrophied rat heart.

STUDY OBJECTIVE--The aim was to determine if the adaptational process of the cardiac calcium channel to pressure overload observed in rat heart also occurs in species characterised by a higher sensitivity to external calcium than in the rat. This adaptation occurs via a maintained density of dihydropyridine receptors and calcium current in hypertrophied rat heart. DESIGN--The guinea pig was chosen and the dissociation constant (Kd), association and dissociation rate constants (k+1,k-1), and maximal number (Bmax) of the dihydropyridine receptors were measured through binding of [3H]PN 200-110 to crude sarcolemma fractions from control and hypertrophied guinea pig left ventricle. EXPERIMENTAL MATERIAL--Hypertrophy of the left ventricle was obtained by stenosis of the abdominal aorta in guinea pigs. MEASUREMENTS AND MAIN RESULTS--Hypertrophy reached at least 50% in 15% of the surviving animals. No significant differences in the binding of [3H]PN 200-110 to the dihydropyridine receptor were observed between control and hypertrophied left ventricle microsomal preparations: Kd = 1.59(SEM 0.22) and 1.17(0.36) nM; Bmax = 225(18) and 213(4) fmol.mg-1 of protein; k-1 = 2.30(0.26) and 2.00(0.13) min-1 x 10(-2); k+1 = 3.8(0.7) and 3.5(0.3) nM-1.min-1 x 10(-2) respectively. CONCLUSIONS--In guinea pig as in rat, the total number of dihydropyridine receptors per left ventricle increased proportionately to the hypertrophy. This is consistent with an unchanged density of the cardiac Ca2+ channels in the hypertrophied guinea pig heart as previously shown in hypertrophied rat heart.     

Properties of lysosomes in guinea pig heart: subcellular distribution and in vitro stability.

Some 20 hydrolytic enzymes, known to be of lysosomal origin in rat liver, were assayed in fractions of guinea pig heart. The enzymes included glycosidases, esterases, proteases (cathepsins) and nucleases. All exhibited structure-linked latency and had optimal activity in acid pH, conforming to the biochemical definition of lysosomal enzymes. Six enzymes which had high activity in guinea pig heart were also measured in rat liver using the fractionation and assay methods employed for myocardium. The acid hydrolase activities of the two tissues are compared and discussed in relation to the cellular origins of the organelles with which they are associated.A lysosome-rich suspension was prepared from guinea pig left ventricle by differential centrifugation. The isolated lysosomes were subjected to various labilizing conditions and the rate of lysosome disruption was monitored by measurements of latent acid hydrolase activity. Loss of latent activity was accelerated by heating, hypotonicity, changes in pH and by addition of NaCl and KCl. The kinetics of lysosomal enzyme release were similar for each of the enzymes tested, with the exception of β-glucuronidase which was released more rapidly by heating and less rapidly by hypotonicity.Subcellular distribution profiles of the acid hydrolases in guinea pig heart were obtained by sucrose density gradient centrifugation. Hearts from both normal and Triton WR 1339 treated animals were fractionated. The modal equilibrium densities of the acid hydrolases revealed the presence of five biochemically distinct lysosomal populations in ventricle tissue, three of which were accessible to Triton WR 1339.The findings in this study show that myocardial lysosomes are a heterogeneous group of organelles with different enzymatic constituents and different physical properties. In view of this there is a need for careful choice of representative marker enzymes in studies on the functions of myocardial lysosomes and their role in disease.     

Geometry of capillary networks in hypertrophied rat heart.

Capillary geometry was examined in normal and hypertrophic myocardium. Hypertrophy was induced by aortic constriction in neonatal rats. Morphometric data were obtained from tissue sections exposed to a staining technique that distinguished the arteriolar and venular portions of capillaries by color. In sham-operated controls, the theoretical tissue region supplied by a single capillary decreased from the arteriolar to venular side (499 +/- 3 microns 2 and 456 +/- 5 microns 2, P less than 0.05; mean +/- SE) of capillaries. In hypertrophy, only arteriolar capillary tissue regions increased in size, thus enlarging the difference between arteriolar and venular ends (547 +/- 6 microns 2 and 464 +/- 5 microns 2, P less than 0.01). Intercapillary distances, measured at various levels along the capillary path length, decreased in a stepwise manner in both normal and hypertrophic hearts. In hypertrophic hearts, mean capillary path length was significantly longer than in controls, but the total length of the individual capillary nets was reduced. In both groups, arteriolar capillary segment length was longer (P less than 0.01) than venular capillary segment length. Given that PO2 values are lower on the venular side of capillaries, this spatially distinctive geometry in normal myocardium: smaller domains, shorter intercapillary distances and segment lengths, would provide favorable geometric conditions for oxygen diffusion. In hypertrophy, average intercapillary distance increased, and the distinction between arteriolar and venular portions of capillaries was further exacerbated.     

Reduced number of digitalis receptor sites in the hypertrophied pig myocardium

Summary Left ventricular hypertrophy was established in young pigs (35–40 kg) after 4 weeks of supravalvular aortic stenosis. Isolated sarcolemmal membrane fraction was characterized for specific³H-ouabain binding. Analysis revealed a single class of receptor sites of 5.7 p moles/mg protein and a dissociation constant (Kd) of the order of 6.25×10^–8 M. Measurement of specific³H-ouabain binding with sarcolemmal fraction isolated from hypertrophied heart revealed a 26% reduction of digitalis receptor after 4 weeks of aortic stenosis, without any significant change in the affinity of receptor sites. This change in the number of digitalis receptors was maintained between 4 and 8 weeks of aortic stenosis and may be an important adaptive mechanism.This investigation was supported by a grant from the Medical Research Council of Canada     

The mechanical characteristics of hypertrophied rabbit cardiac muscle in the absence of congestive heart failure: the contractile and series elastic elements.

Right ventricular papillary muscles from normal rabbits and rabbits with sustained pulmonary artery constriction (67% decrease in external diameter) were studied at several resting muscle lengths and at an early instant in the isometric twitch. Instantaneous force-velocity data were obtained at 30-38% of time to peak tension (TPT) and at 96%, 98%, and 100% of the resting muscle length at which active twitch tension was maximal. Unloaded shortening velocity (Vmax) was estimated with a linearized form of the Hill hyperbolic formula, and was depressed in hypertrophy to 36% less than normal. We found that Vmax did not change with muscle length in the normal or hypertrophied muscles; therefore there was a length- and time-independent depression of contractile element shortening capacity that was consistent with previous work from this laboratory which demonstrated a depression of myosin and actomyosin ATPase activity in hypertrophy.     

Abnormalities of calcium cycling in the hypertrophied and failing heart

Progressive deterioration of cardiac contractility is a central feature of congestive heart failure (CHF) in humans. In this report we review those studies that have addressed the idea that alterations of intracellular calcium (Ca(2+)) regulation is primarily responsible for the depressed contractility of the failing heart. The review points out that Ca(2+)transients and contraction are similar in non-failing and failing myocytes at very slow frequencies of stimulation (and other low stress environments). Faster pacing rates, high Ca(2+)and beta-adrenergic stimulation reveal large reductions in contractile reserve in failing myocytes. The underlying cellular basis of these defects is then considered. Studies showing changes in the abundance of L-type Ca(2+)channels, Ca(2+)transport proteins [sarcoplasmic reticulum Ca(2+)ATPase (SERCA2), phospholamban (PLB), Na(+)/Ca(2+) exchanger (NCX)] and Ca(2+) release channels (RYR) in excitation-contraction coupling and Ca(2+)release and uptake by the sarcoplasmic reticulum (SR) are reviewed. These observations support our hypotheses that (i) defective Ca(2+)regulation involves multiple molecules and processes, not one molecule, (ii) the initiation and progression of CHF inolves defective Ca(2+)regulation, and (iii) prevention or correction of Ca(2+)regulatory defects in the early stages of cardiac diseases can delay or prevent the onset of CHF.