Correlation between endogenous polyamines in human cardiac tissues and clinical parameters in patients with heart failure

Polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy in experimental animals. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with cyclic adenosine monophosphate (cAMP) increases. The aim of the study was to establish the role of these in the human heart in living patients. For this, polyamines (by high performance liquid chromatography) and the activity of ODC and N¹‐acetylpolyamine oxidases (APAO) were determined in the right atrial appendage of 17 patients undergoing extracorporeal circulation to correlate with clinical parameters. There existed enzymatic activity associated with the homeostasis of polyamines. Left atria size was positively associated with ODC (r = 0.661, P = 0.027) and negatively with APAO‐N¹‐acetylspermine (r = −0.769, P = 0.026), suggesting that increased levels of polyamines are associated with left atrial hemodynamic overload. Left ventricular ejection fraction (LVEF) and heart rate were positively associated with spermidine (r = 0.690, P = 0.003; r = 0.590, P = 0.021) and negatively with N¹‐acetylspermidine (r = −0.554, P = 0.032; r = −0.644, P = 0.018). LVEF was negatively correlated with cAMP levels (r = −0.835, P = 0.001) and with cAMP/ODC (r = −0.794, P = 0.011), cAMP/spermidine (r = −0.813, P = 0.001) and cAMP/spermine (r = −0.747, P = 0.003) ratios. Abnormal LVEF patients showed decreased ODC activity and spermidine, and increased N¹‐acetylspermidine, and cAMP. Spermine decreased in congestive heart failure patients. The trace amine isoamylamine negatively correlated with septal wall thickness (r = −0.634, P = 0.008) and was increased in cardiac heart failure. The results indicated that modifications in polyamine homeostasis might be associated with cardiac function and remodelling. Increased cAMP might have a deleterious effect on function. Further studies should confirm these findings and the involvement of polyamines in different stages of heart failure.     

Age- and myopathy-dependent changes in connexins of normal and cardiomyopathic Syrian hamster ventricular myocardium

The conduction of cardiac action potentials depends on the flow of excitation through gap junctions, which are hexameric protein associations of connexins (Cxs). The major Cx reported in the heart is Cx43, although some Cx40 and Cx45 are also present. There is some evidence for altered Cx content in heart failure. In heart failure, conduction is depressed and slowed conduction may contribute to arrhythmogenesis and (or) the maintenance of arrhythmia. Cx content and distribution were determined in ventricular tissues from normal and cardiomyopathic Syrian hamsters, an animal model of heart failure which has reproducible age-specific cardiomyopathy resulting in heart failure and age-matched controls in three groups: young (3-5 weeks), adult (13-18 weeks), and old (>45 weeks). Frozen, unfixed sections of ventricular tissues were immunofluorescently stained using antibodies against Cx43, Cx40, and Cx45. Cx43 was the predominant Cx detected in all samples. In normal hamsters, Cx43 was localized predominantly at the intercalated disc region, while in myopathic myocytes, it was scattered. In Western blots, Cx43 content of normal hamster hearts was highest in the adult hearts compared with young and old hamster hearts. In contrast, Cx43 content was significantly lower in adult cardiomyopathic hamster hearts compared with all other groups. The alterations of content and distribution of gap junction Cx43 may contribute to diminished conduction, pump function, and arrhythmogenesis in heart failure.     

Glycogen phosphorylase activities in skeletal muscle and heart of genetically dystrophic Syrian hamster.

A simple, rapid and reliable procedure of tissue preparation was devised to estimate glycogen phosphorylase activity in cardiac and skeletal muscle of normal and genetically dystrophic Syrian hamsters of various ages. Total phosphorylase activities of dystrophic skeletal muscle, compared to normal, were reduced. Except for the case of heart from the younger dystrophic animals (45 days old), in which higher phosphorylase activity was noted, hearts from dystrophic hamsters, compared to normal, also showed reduced phosphorylase activities. There were, however, no significances in the ratios of phosphorylase alpha to total phosphorylase between the normal and dystrophic tissues.     

Epicardial adipose tissue assessed by cardiac magnetic resonance imaging in patients with heart failure due to dilated cardiomyopathy

Objective:

We sought to investigate the association of the EAT with CMR parameters of ventricular remodelling and left ventricular (LV) dysfunction in patients with non‐ischemic dilated cardiomyopathy (DCM).

Design and Methods:

One hundred and fifty subjects (112 consecutive patients with DCM and 48 healthy controls) underwent CMR examination. Function, volumes, dimensions, the LV remodelling index (LVRI), the presence of late gadolinium enhancement (LGE) and the amount of EAT were assessed.

Results:

Compared to healthy controls, patients with DCM revealed a significantly reduced indexed EAT mass (31.7 ± 5.6 g/m² vs 24.0 ± 7.5 g/m², p<0.0001). There was no difference in the EAT mass between DCM patients with moderate and severe LV dysfunction (23.5 ± 9.8 g/m² vs 24.2 ± 6.6 g/m², P = 0.7). Linear regression analysis in DCM patients showed that with increasing LV end‐diastolic mass index (LV‐EDMI) (r = 0.417, P < 0.0001), increasing LV end‐diastolic volume index (r = 0.251, P = 0.01) and increasing LV end‐diastolic diameter (r = 0.220, P = 0.02), there was also a significantly increased amount of EAT mass. However, there was no correlation between the EAT and the LV ejection fraction (r = 0.0085, P = 0.37), right ventricular ejection fraction (r = 0.049, P = 0.6), LVRI (r = 0.116, P = 0.2) and the extent of LGE % (r = 0.189, P = 0.1). Among the healthy controls, the amount of EAT only correlated with increasing age (r = 0.461, P = 0.001), BMI (r = 0.426, P = 0.003) and LV‐EDMI (r = 0.346, P = 0.02).

Conclusion:

In patients with DCM the amount of EAT is decreased compared to healthy controls irrespective of LV function impairment. However, an increase in LV mass and volumes is associated with a significantly increase in EAT in patients with DCM.     

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.     

Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure

Summary Purkinje fibers of the sheep heart were exposed to (a) 0.1mm dihydro-ouabain (DHO), followed by (b) 0.1mm DHO in Na-free solution or to (c) 1mm dinitrophenol (DNP). The degree of electrical decoupling was characterized in terms of the inside longitudinal resistancer _i as measured with a 3-microelectrode voltage-clamp technique. Procedurea increasedr _i by a factor of 3.7±1.1 (mean±sd),b by a factor of 9.8±2.2, whereas inc incomplete voltage control indicated nearly complete uncoupling. Intracellular calcium activity (aCa _i ) was monitored with a microelectrode system. At control conditionsaCa _i was below 0.1 m. The procedures listed above increasedaCa _i to (a) 4±1.5 m, (b) 8±2 m, and (c) 36±12 m. The increase ofaCa _i was in good correlation with the changes in core resistance. Effects on nexus ultrastructure, investigated with freeze-fracture techniques, are shown in histograms. At control conditions, the particle diameter distributed around a single peak (8.3±0.5 nm). Proceduresb andc induced a second population at 10.8 nm; increased decoupling reduced the control population in favor of the 10.8 nm population. Decoupling enlarged the width of the nexus gap by a factor of 1.6; again, the control population decreased in favor of a new population. In the decoupled state the height of the particle was smaller. Pits on the E-face displayed a more regular array and a nearly unchanged center-to-center spacing. Separation into several peaks was not possible due to scatter of the data.We interpret the findings to mean that elevatedaCa _i induces a conformational change of the nexus subunits which corresponds to a transition from an open to a closed state. The conformational change can be formally described by a particle contraction which disrupts the continuity with the particle of the adjacent membrane. Purkinje fibers exposed to DNP for 1 hr showed thinned (7.7±0.5 mm) and elongated particles. We suggest that this is a secondary event and not a precursor of functional uncoupling.     

Reduced cardiac myofibrillar Mg-ATPase activity without changes in myosin isozymes in patients with end-stage heart failure

In this study we tested the hypothesis that reduced myofibrillar ATPase activities in end-stage heart failure are associated with a redistribution of myosin isozymes. Cardiac myofibrils were isolated from left ventricular free wall from normal human hearts and hearts at end-stage heart failure caused by coronary artery diseases, cardiomyopathy or immunological rejection. The hearts had been excised in preparation for a heart transplant. Myofibrillar Ca^2–-dependent Mg-ATPase and myosin Ca^–- and K^–EDTA-ATPase activities were compared. Possible changes in myosin isozyme distribution in the diseased heart were investigated using polyacrylamide gel electrophoresis of native myosin in the presence of pyrophosphate. Significant reduction in myofibrillar Ca²⁺-dependent Mg-ATPase with no changes in the sensitivity of the myofibrils to Ca⁺ was observed in heart with coronary artery diseases (25.2 to 27.1% at pCa 5.83 to pCa 5.05), cardiomyopathy (21.1 to 25.5% at pCa 5.41 to pCa 5.05), and in the immunologically rejected heart (18.4 to 22.8% at pCa 5.41 to pCa 5.05). Significantly lower myosin Ca²⁺-ATPase was observed with coronary artery diseases only and myosin K-EDTA activities did not differ in diseased and normal hearts. Polyacrylamide gel electrophoresis of native myosin from the normal and three models of end-stage heart failure revealed two distinct bands in the human left ventricle and one diffuse band in the human right atria. No apparent differences in myosin isoenzyme pattern were observed between the normal and diseased hearts. Further evaluation is needed to clarify the ATPase nature of the two bands.     

Role of ion channels in heart failure and channelopathies

Heart failure (HF) is a complication of multiple cardiac diseases and is characterized by impaired contractile and electric function. Patients with HF are not only limited by reduced contractile function but are also prone to life-threatening ventricular arrhythmias. HF itself leads to remodeling of ion channels, gap junctions, and intracellular calcium handling abnormalities that in combination with structural remodeling, e.g., fibrosis, produce a substrate for an arrhythmogenic disorders. Not only ventricular life-threatening arrhythmias contribute to increased morbidity and mortality but also atrial arrhythmias, especially atrial fibrillation (AF), are common in HF patients and contribute to morbidity and mortality. The distinct ion channel remodeling processes in HF and in channelopathies associated with HF will be discussed. Further basic research and clinical studies are needed to identify underlying molecular pathways of HF pathophysiology to provide the basis for improved patient care and individualized therapy based on individualized ion channel composition and remodeling.     

Mutations of presenilin genes in dilated cardiomyopathy and heart failure

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.     

Sodium-conducting channels in cardiac membranes in low calcium.

With no Ca in the patch electrode, two kinds of channels conduct Na in spontaneously beating embryonic chick heart cells. One channel conducts Na primarily during the upstroke of the action potential and is blocked by tetrodotoxin (TTX). The other channel conducts Na primarily during the late plateau and early repolarization phase of the action potential, but only in Ca concentrations below 10(-6) M. This second channel is TTX-insensitive and has a conductance of 50 to 90 pS, depending upon the interpretation of open-channel flickering. These two Na-conducting channels correspond to the channels that normally carry the fast Na current (INa) and the slow Ca current (Isi).     

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.     

Hormonal regulation of membrane calcium channels in the heart

It has been shown on the trabecules of frog auricle by potential recording, that prostaglandin E1 (PGE1) increases the number of working calcium channels of the membrane. PGE1, PGE2, acetylcholine, indometacin--inhibitor of PG synthesis and aminazine 2--calmodulin inhibitor do not decrease novodrin--induced intensification of calcium current. The results suggest that a change of cAMP level inside the cell modulates the number of working calcium channels. PG and calmodulin are not necessary for novodrin--stimulation of adenylatcyclase (AC). The effect of acetylcholine is not connected with AC inhibition.     

Two polypeptide changes associated with butyric acid resistance and the neoplastic state of Syrian hamster cells.

Seven differences in the polypeptide species of parental Syrian hamster embryo cells and cells of the highly tumorigenic derivative cell line BP6T were identified previously by employing the technique of two-dimensional polyacrylamide gel electrophoresis (Leavitt, J. and Moyzis, R. (1978) J. Biol. Chem. 253, 2497-2500). To determine which of these polypeptide changes are correlated with expression of the neoplastic state this work was extended to the comparative examination of nine established neoplastic cell lines which resulted from independent transformation events catalyzed by chemical carcinogen treatment, virus infection, or an unknown spontaneous event. Although no perfect correlation with a specific polypeptide change was found, two polypeptide changes, occurring independently or simultaneously, appear to be consistently associated with expression of neoplasticity. One polypeptide species, designated tau, having an isoelectric point of 4.6 and a molecular weight of 60 000 was lost or physically altered in all but one of these transformed cell lines; a second polypeptide species designated nu having an isoelectric point 5.5 and a molecular weight of 42 000 appeared in highly tumorigenic chemically transformed cell lines and in two virally transformed cell lines. A butyric acid supplement, used as a selective agent for butyric acid resistant cells, was employed to identify and isolate in a single step nascent neoplastic clonal lines transformed by ethylmethanesulfonate. These cell lines exhibited alterations either in tau or nu. The changes observed in tau are consistent with those expected to result from a somatic mutation event in the structural gene coding for tau; however, the alterations in tau could also be governed by a post-translational process. These findings suggest that alterations in expression of at least two major polypeptide species, tau and nu, are closely associated with primary steps in the neoplastic transformation process of Syrian hamster cells irrespective of the nature of the transforming agent.     

Kinetic changes of ethanolamine base exchange activity and increase of viscosity in sarcolemmal membranes of hamster heart during development of cardiomyopathy

The activity of the phospholipid base exchange enzyme specific for ethanolamine has been measured in cardiac sarcolemmal membrane preparations from Syrian golden and UM-X7.1 cardiomyopathic hamsters. In Syrian golden hamsters, the Km of the enzyme for ethanolamine does not change with age, whereas it almost doubles in membranes from cardiomyopathic animals, from the 30th to the 150th day of age. During the same period, the membrane cholesterol content increases by 68% in cardiomyopathic hamsters, whereas it does not change significantly in the Syrian golden hamster strain. As a consequence, in the adult animal, the cholesterol to phospholipid ratio and the viscosity of sarcolemmal membranes are higher in UM-X7.1 strain than in Syrian golden hamsters. A cause consequence relationship between the enzymatic changes and the compositional modifications in the sarcolemma occurring in UM-X7.1 hamsters during the development of cardiomyopahhy is proposed. (Mol Cell Biochem 116: 89–93,1992)     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.