Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca²⁺ movements and the association of abnormal Ca²⁺ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.     

Losartan corrects abnormal frequency response of renal vasculature in congestive heart failure

In congestive heart failure, renal blood flow is decreased and renal vascular resistance is increased in a setting of increased activity of both the sympathetic nervous and renin-angiotensin systems. The renal vasoconstrictor response to renal nerve stimulation is enhanced. This is associated with an abnormality in the low-pass filter function of the renal vasculature wherein higher frequencies (> or =0.01 Hz) within renal sympathetic nerve activity are not normally attenuated and are passed into the renal blood flow signal. This study tested the hypothesis that excess angiotensin II action mediates the abnormal frequency response characteristics of the renal vasculature in congestive heart failure. In anesthetized rats, the renal vasoconstrictor response to graded frequency renal nerve stimulation was significantly greater in congestive heart failure than in control rats. Losartan attenuated the renal vasoconstrictor response to a significantly greater degree in congestive heart failure than in control rats. In control rats, the frequency response of the renal vasculature was that of a first order (-20 dB/frequency decade) low-pass filter with a corner frequency (-3 dB, 30% attenuation) of 0.002 Hz and 97% attenuation (-30 dB) at > or =0.1 Hz. In congestive heart failure rats, attenuation did not exceed 45% (-5 dB) over the frequency range of 0.001-0.6 Hz. The frequency response of the renal vasculature was not affected by losartan treatment in control rats but was completely restored to normal by losartan treatment in congestive heart failure rats. The enhanced renal vasoconstrictor response to renal nerve stimulation and the associated abnormality in the frequency response characteristics of the renal vasculature seen in congestive heart failure are mediated by the action of angiotensin II on renal angiotensin II AT1 receptors.     

Pacing in congestive heart failure

Despite the major advances in medical drug therapy, heart failure remains a syndrome associated with high mortality and morbidity. Biventricular or left ventricular (LV) short atrioventricular (AV) delay pacing is being tested in congestive heart failure patients with left bundle branch block. The aim is to resynchronise the dyscoordinate LV contraction. A number of studies are underway, but it is clear that while some patients respond remarkably, this is highly variable. Accurate identification of patients likely to benefit will be crucial. The mechanism of benefit is unclear. A greater understanding of the physiological consequences of pacing will be necessary to accurately identify these patients.     

A new model of congestive heart failure in rats

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Alterations in G-proteins in congestive heart failure in cardiomyopathic (UM-X7.1) hamsters

In order to explain the attenuated sympathetic support during the development of heart failure, the status of -adrenergic mechanisms in the failing myocardium was assessed by employing cardiomyopathic hamsters (155–170 days old) at moderate degree of congestive heart failure. The norepinephrine turnover rate was increased but the norepinephrine content was decreased in cardiomyopathic hearts. The number and the affinity of receptors in the sarcolemmal preparations were not changed in these hearts at moderate stage of congestive heart failure. While the basal adenylyl cyclase activity was not altered in sarcolemma, the stimulation of enzyme activity by NaF, forskolin, Gpp(NH)p or epinephrine was depressed in hearts from these cardiomyopathic hamsters. Since G-proteins are involved in modifying the adenylyl cyclase activity, the functional and bioactivities as well as contents of both Gs and Gi proteins were determined in the cardiomyopathic heart sarcolemma. The functional stimulation of adenylyl cyclase by cholera toxin, which activates Gs proteins, was markedly depressed whereas that by Pertussis toxin, which inhibits Gi proteins, was markedly augmented in cardiomyopathic hearts. The cholera toxin and pertussis toxin catalyzed ADP-ribosylation was increased by 37 and 126%, respectively; this indicated increased bioactivities of both Gs and Gi proteins in experimental preparations. The immunoblot analysis suggested 74 and 124% increase in Gs and Gi contents in failing hearts, respectively. These results suggest that depressed adenylyl cyclase activation in cardiomyopathic hamsters may not only be due to increased content and bioactivity of Gi proteins but the functional uncoupling of Gs proteins from the adenylyl cyclase enzyme may also be involved at this stage of heart failure.     

Dipalmitoylphosphatidylcholine (L-alpha-lecithin) stimulates phospholipase A2 activity in human amnion

In order to investigate the mechanism of dipalmitoylphosphatidylcholine (DPPC, L-alpha-lecithin) stimulation of the prostaglandin E (PGE) production of the amniotic membrane, effects of DPPC (50-800 micrograms/ml) on phospholipase A2 (PLA2), phospholipase C (PLC), PG endoperoxide synthase, and PGE synthase activities of human amniotic membrane were studied. Only PLA2 activity was increased by DPPC, suggesting that lecithin, the major surfactant component, increases the PGE production of the amniotic membrane by activating PLA2.     

Role of endothelins in congestive heart failure

Despite major advances in conventional medical therapy, patients with heart failure continue to experience significant morbidity and mortality. Endothelin-1 (ET-1) is a potent vasocontrictor and mitogenic peptide that is activated in heart failure. There is increasing experimental and clinical evidence in support of an important role of ET-1 in the pathophysiology of heart failure. Manipulation of the activity of ET-1, especially using endothelin receptor blockers, has allowed for the further elucidation of the role of this neurohormonal system and development of novel therapeutic strategies in heart failure. Published clinical studies of these agents to date have involved relatively small numbers of patients with severe heart failure, followed for a relatively short period of time, and have mainly examined surrogate endpoints. Large-scale trials that address to hard clinical outcomes are ongoing and their results forthcoming. A key question that remains concerns whether selective ETA or dual ETA-ETB receptor blockade will be more effective.     

Alpha 1-adrenoceptor activity in arterial smooth muscle following congestive heart failure

The interactions between yohimbine (selective alpha 2-antagonist) with noradrenaline (mixed agonist) and phenylephrine (selective alpha 1-agonist) were studied in the canine dorsal pedal artery in an attempt to characterize the peripheral vascular response to adrenergic agents before and after the development of congestive heart failure in the dog. The contractile responses of the dorsal pedal artery to potassium chloride were also examined. Both noradrenaline and phenylephrine contracted the dorsal pedal artery in a concentration-dependent manner before and at peak heart failure, the responses to the agonists being enhanced at heart failure. The responses of the artery to potassium were not modified by congestive heart failure. Yohimbine caused concentration-dependent antagonism of noradrenaline, without altering the magnitude of the maximum response, providing pA2 values ranging from 8.26 to 7.06 against low and high concentrations of noradrenaline, respectively, before heart failure development. Following heart failure, the pA2 values for yohimbine against noradrenaline remained unchanged, but slopes from the Arunlakshana-Schild plots were significantly different from unity, implying a noncompetitive antagonism. The pA2 values of yohimbine against phenylephrine were at least 10 orders of magnitude lower than those against noradrenaline. After congestive heart failure, yohimbine was even less effective against high concentrations of phenylephrine. These findings suggest that enhanced vasoconstriction during heart failure results, in part, from increased alpha 1-adrenoceptor mechanisms in peripheral arterial smooth muscle.     

Enhanced matrix metalloproteinase activity in skeletal muscles of rats with congestive heart failure

Patients with congestive heart failure (CHF) are prone to increased skeletal muscle fatigue. Elevated circulatory concentrations of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1, which may stimulate matrix metalloproteinase (MMP) activity and, thereby, contribute to skeletal muscle dysfunction, are frequently found in CHF. However, whether skeletal muscle MMP activity is altered in CHF is unknown. Hence, we have used a gelatinase assay to assess the activity of MMP and tissue inhibitors of MMP in single skeletal muscles of rats with CHF 6 wk after induction of myocardial infarction. Sham-operated (Sham) rats were used as controls. We also measured the gene expression and protein contents of MMP-2 and MMP-9 in skeletal muscles of these rats. Plasma MMP activity was nearly seven times higher (P < 0.05) in CHF than in Sham rats. Concomitantly, the MMP activity within single slow- and fast-twitch skeletal muscles of CHF rats increased two- to fourfold compared with Sham animals, whereas tissue inhibitor of MMP activity did not differ (P > 0.05). Preformed MMP-2 and MMP-9 were probably activated in CHF, because neither their gene expression nor protein levels were altered (P > 0.05). Serum concentrations of TNF-alpha and monocyte chemoattractant protein-1 remained unchanged (P > 0.05) between CHF and Sham rats during the 6-wk observation period. We conclude that development of CHF in rats enhances MMP activity, which in turn may distort the normal contractile function of skeletal muscle, thereby contributing to increased skeletal muscle fatigue.     

Cheyne-Stokes respiration in congestive heart failure.

Cheyne-Stokes respiration is an abnormal breathing pattern which commonly occurs in patients with decompensated congestive heart failure and neurologic diseases, in whom periods of tachypnea and hyperpnea alternate with periods of apnea. In the majority of these patients, the ventilatory patterns may not be recognized, and the clinical features are generally dominated by the underlying disease process. Cheyne-Stokes respiration may, however, have profound effects on the cardiopulmonary system, causing oxygen desaturation, cardiac arrhythmias, and changes in mental status. Treatment of Cheyne-Stokes respiration in congestive heart failure with supplemental oxygen or nasal continuous positive airway pressure, in addition to conventional therapy, may improve the overall cardiac function and perhaps the patient's prognosis.     

Characterization of cockroach (Periplaneta americana) fat body phospholipase A(2) activity

A phospholipase has been identified in the fat body of the American cockroach, Periplaneta americana, which removes fatty acid from the sn-2 acyl position of an artificial substrate. The enzyme has been characterized using a crude preparation obtained by low-speed centrifugation of the homogenized tissue. With 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine as the substrate, the K(m) has been estimated to be 1.17 microM and the v(max) 113.5 pmol/min/mg protein. The phospholipase has a pH optimum close to 7 and shows maximal activity at 50 degrees C. Activity of the phospholipase has been determined in cytosolic and plasma membrane fractions. The specific activity of the latter fraction is approximately twice that of the cytosol. The enzyme in both fractions is Ca(2+)-independent. Arch.     

The role of calcium-independent phospholipase A2 in cardiolipin remodeling in the spontaneously hypertensive heart failure rat heart

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)₄CL]. A decrease in (18:2)₄CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)₄CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A₂ (iPLA₂) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)₄CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA₂ inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)₄CL in the failing rat heart, and that mitochondrial iPLA₂ plays a role in the remodeling of CL''s acyl composition.