Calcium transport by intracellular membrane structures in the myocardium of hypertrophied and failing hearts

After inducing haemodynamic cardiac overload in rabbits, the authors studied in several stages (1-14 months) the calcium transport activity of the mitochondrial and sarcoplasmic myocardial fractions using labelled 45CaCl2. A coincidence was found between changes in myocardial contractility and changes in calcium transport activity of intracellular organelles. A possible important role of mitochondria in this adaptive process was also documented. Since the calcium transport capacity of the sarcoplasmic reticulum progressively decreases (with the exception of the earliest stages following overload induction), it seems that increased myocardial contractility ensures enhanced Ca transport activity of the mitochondria. Myocardial contractility drops only at the time when the Ca transport activity of the mitochondria decreases. Since these changes occur already at the time of regression of myocardial hypertrophy, which precedes heart failure, it can be assumed that they are causally connected with the reduced contractility of a failing heart.     

Calcium immobilization in mitochondria of failing myocardium: X-ray microanalytical evidence

Electron microscopic study of myocardial cells from rat failing heart (chronic congestive heart failure—advanced, near-terminal state—produced by stenosis of the descending aorta) showed that about 80% of mitochondrial profiles contained electron-opaque deposits, which occupied about 5% of the mitochondrial volume. X-ray analytical electron microscopy of mitochondria in situ (line-scanning and spot analyses) indicated that these electron-opaque deposits contained a significant amount of calcium. The mitochondrial electron-opaque deposits represent sites of calcium immobilization in a complex of proteins and lipids, presumably derived from damaged mitochondrial membranes.     

Calcium transport and phospholamban in sarcoplasmic reticulum of ischemic myocardium

The present study was designed to examine the relation between the loss of Ca2+ uptake activity and the change of protein phosphorylation in sarcoplasmic reticulum from ischemic myocardium. Ischemic (0.5, 1 and 2 h duration) and non-ischemic tissue samples were taken from the coronary-ligated porcine left ventricle and sarcoplasmic reticulum fractions were isolated. The membranes were tested for Ca2+ uptake and ATPase activities and phosphorylation of phospholamban. The in vitro 32P incorporation into phospholamban in the presence of cAMP plus the catalytic subunit of cyclic AMP dependent protein kinase became markedly reduced depending on the duration of ischemia. The activities of the Ca2+ pump (Ca2+ uptake and ATPase) were also decreased. The 32P incorporation into the myofibrillar component troponin I, which is also a specific substrate for catalytic subunit, was not affected by ischemia. The reduction of the Ca2+ pump activity correlated with the reduction of 32P incorporation into phospholamban. It is postulated that the ischemia induced inactivation of the Ca2+ pump is not only a consequence of specific loss of enzyme activity, but it is also caused by altered characteristics of phospholamban.     

Inotropic therapy for the failing myocardium

Congestive heart failure (CHF) is a common clinical entity with diverse causes. Patients may present with acute decompensation or have a more indolent course, with diminishing exercise tolerance and increasing dyspnea. The management of this clinical entity traditionally has focused on restricting fluid intake, decreasing dietary sodium, decreasing afterload with vasodilatory agents, and supporting the failing myocardium with agents which produce a positive inotropic response. In the acutely decompensated patient, short-term therapy with positive inotropics is clearly beneficial. The role of long-term inotropic therapy for chronic CHF remains less clear. A number of clinical trials have recently evaluated the effects of long-term therapy on morbidity and mortality, with disappointing results. For a number of the newer, nonglycocide oral positive inotropics, at doses of drug which produce measurable hemodynamic improvement, increased mortality in treatment groups has been unacceptably high. Ironically, patients with worse left ventricular dysfunction show the most clinical improvement, but have the highest increased mortality. However, as with digoxin, there is some evidence that employing lower doses of drug that do not produce measurable improvement in hemodynamic parameters may result in both improved clinical state and decreased mortality. This review discusses the role of both oral and intravenous inotropic agents, discusses the difficulty in translating short-term hemodynamic improvement into long-term clinical benefit, and presents a rationale for the use of lower-dose inotropic therapy to improve long-term clinical outcome.     

Inotropic therapy of the failing myocardium

The clinical syndrome of congestive heart failure remains a therapeutic dilemma and challenge for the physician in 1992. This is a disease process that appears to be increasing in frequency and continues to carry an unacceptably high mortality rate. For years it has been well recognized that the combination of digoxin, Lasix and vasodilator therapy improved symptoms in these patients and decreased hospitalization, but did not increase survival. It was not until 1986 that the combination of digoxin, Lasix, Isordil, and hydralazine was shown to increase survival. Further significant improvement in quality of life and survival has recently been established in three large clinical trials, and it is now safe to say that the standard of care for symptomatic congestive heart failure in 1992 is digoxin, furosemide, and an ACE inhibitor, with the survival trials favoring the ACE inhibitor enalapril. The IV inotropic drug dobutamine remains the mainstay of pharmacological therapy for the treatment of severely refractory heart failure. Unfortunately, the phosphodiesterase inhibitors—amrinone, milrinone, and enoximone—have demonstrated unacceptable clinical side effects and have been withdrawn from further clinical study. In spite of these promising developments, the mortality and morbidity of congestive heart failure remains unacceptably high, and continued investigation in the new fields of pharmacology and the pathophysiology of congestive heart failure still must be aggressively pursued.     

Diminished posttetanic potentiation in failing human myocardium

In heart failure a decreased function of SERCA2 has been demonstrated. The present study aimed at investigating the relation between sarcoplasmic reticulum-Ca²⁺-load (SR-Ca²⁺-load) and the activity of the SERCA2. SR-Ca²⁺ load was evaluated by measuring posttetanic potentiation (PTP) in human nonfailing (NF, n=10) and endstage failing myocardium (DCM, n=11). In addition, the effect of cyclopiazonic acid (CPA), a specific inhibitor of SERCA2, on PTP was studied in both NF and DCM. In crude membrane preparations from the same hearts the maximal SERCA2 activity was determined and correlated with the PTP. In failing myocardium the PTP was significantly reduced compared to nonfailing myocardium (13.7±0.75 mN/mm² vs. 17.1±1.55 mN/mm², p<0.05, ±SEM). When PTP was studied in the presence of increased extracellular Ca²⁺-concentrations, the difference between NF and DCM was further pronounced. CPA decreased PTP in both nonfailing and failing human tissue. The maximal SERCA2 activity was significantly reduced in failing myocardium (NF 267±18.5 nmol ATP/mg protein · min⁻¹ vs. DCM 191±13.4 nmol ATP/mg protein · min⁻¹, p<0.05, ± SEM). Correlation of the PTP and maximal SERCA2 activity revealed a close correlation between both parameters in NF and DCM. In summary, the presented results suggest that reduced SERCA2 activity in DCM influences posttetanic force potentiation probably through a reduced SR-Ca²⁺-load. Received: 30 July 1999 Returned for 1. revision: 9 September 1999 1. Revision received: 24 November 1999 Returned for 2. revision: 26 January 2000 2. Revision received: 26 April 2000 Accepted: 9 May 2000     

Intracellular distribution of adrenoceptors in the failing human myocardium

Although it is increasingly recognized that the density of cardiac membrane-bound beta adrenoceptors declines in heart failure, the mechanisms involved are unclear. Furthermore, it is not known whether cardiac alpha-1 adrenoceptors are similarly affected. Inasmuch as agonist-induced desensitization results in translocation of adrenoceptors from the plasma membrane to an intracellular vesicular fraction, we determined the intracellular distribution of cardiac adrenoceptors in two groups: group 1 (n = 9) consisted of papillary muscles from patients with mild-to-moderate heart failure undergoing valve replacement, and group 2 (n = 8) consisted of severely failing hearts removed during orthotopic cardiac transplantation. The density of cardiac beta adrenoceptors was lower in membranes from group 2 (17.8 +/- 3.3 fmol/mg protein vs 27.8 +/- 3.7 fmol/mg in group 1; (p less than 0.01), and the percentage of beta receptors recovered in the vesicular fraction was higher in group 2 (47.1 +/- 3.3% vs 36.8 +/- 5.0% in group 1; p less than 0.01). In group 1 but not group 2 there was a significant inverse correlation (r = -0.87; p less than 0.001) between the density of membrane-bound beta receptors and the percentage of beta receptors recovered in the vesicular fraction. Alpha-1 adrenoceptors were lower in both membrane and vesicular fraction of group 2 compared to group 1; in group 2 but not group 1 there was a significant negative correlation between the density of membrane-bound alpha-1 adrenoceptors and the percentage of alpha-1 receptors in the vesicular fraction (r = -0.8; p less than 0.01). These results suggest that the regulation of alpha-1 and beta adrenoceptors differs in the failing myocardium. Furthermore, agonist-induced desensitization may play a predominant role only in mild-to-moderate heart failure.     

Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium.

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5, 5'-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274+/-13 versus 136+/-9 nmol. min(-1). mg(-1) protein, P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.     

The effect of xamoterol in failing human myocardium

As the dual pharmacological action of partial beta 1-adrenoceptor agonists should improve left ventricular function while also protecting the myocardium against excessive sympathetic stimulation they may be useful in the treatment of heart failure. Therefore, we studied the pharmacological effects of xamoterol (Corwin, ICI 118, 587), a compound with mixed agonist and antagonistic properties at cardiac beta-adrenoceptors in electrically driven human papillary muscle strips from failing human hearts. Specimens were obtained from patients with different grades of myocardial failure who underwent mitral valve replacement (NYHA II-III) or heart transplantation (NYHA IV). Xamoterol (0.0001-100 mumol l-1) produced only negative inotropic effects, as measured by changes in isometric force of contraction in diseased human papillary muscle strips. However, isoprenaline (0.0001-10 mumol l-1) and ouabain (0.01-0.3 mumol l-1) enhanced force of contraction in the same hearts. Prestimulation with noradrenaline (3 mumol l-1) augmented the negative inotropic effect of xamoterol. It is concluded that xamoterol exerts primarily beta-adrenoceptor antagonistic activity in the failing human myocardium.     

Titin-based mechanical signalling in normal and failing myocardium

Nodal points of mechanotransduction are found along the cardiac sarcomere, notably in the Z-disc/I-band and M-band regions. A major integrating component of these mechanosensitive complexes is the giant protein titin, which is anchored at the Z-disc, spans the I-band as an elastic spring and enters the A-band bound to myosin, then reaching all the way to the M-band. Passive-force generation and transmission of stress via the titin filaments may be central to the mechanosensory function of the myofibrillar signalosome complexes. This review discusses recent findings shedding light on mechanisms by which titin elasticity is regulated dynamically. Adjustment of titin stiffness occurs during heart development and disease through a shift in the expression ratio of the two main titin isoforms in cardiac sarcomeres, N2BA (compliant) and N2B (stiffer). Titin-isoform switching in favor of the stiffer N2B-titin can be triggered by thyroid hormone (T3) activating the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. Conversely, low T3 promotes the compliant N2BA-titin. In addition, titin stiffness can be tuned acutely by protein kinase (PK)A-or PKG-mediated phosphorylation of a cardiac-specific I-band titin segment, the N2-B domain. Beta-adrenergic agonists, nitric oxide, or natriuretic peptides thus trigger a softening of the titin springs, thereby modulating diastolic function. Failing human hearts can have elevated passive stiffness in part because of a titin-phosphorylation deficit, which may contribute to mechanical dysfunction. Altered titin phosphorylation could also affect protein-protein interactions in the mechanosensory complexes associated with the sarcomere. In this context, the review highlights novel links between titin and stress-signalling pathways in the cardiomyocyte.     

Left ventricular assist device-induced molecular changes in the failing myocardium

PURPOSE OF REVIEW: There is considerable increase in the use of left ventricular assist devices for the treatment of severe heart failure. Traditionally viewed as a bridge to transplantation and more recently as a destination therapy, left ventricular assist device support is now recognized to offer potential for myocardial recovery through reverse remodeling, a potential that is further enhanced by combination with pharmacologic therapy. In this study, we examine the molecular changes associated with left ventricular assist device support and how these may contribute to the recovery process. RECENT FINDINGS: Studies in both patients and experimental models have demonstrated that improved function is associated with alterations in several key pathways including cell survival, cytokine signaling, calcium handling, adrenergic receptor signaling, cytoskeletal and contractile proteins, energy metabolism, extracellular matrix, and endothelial and microvascular functions. Moreover, the unique research opportunities offered by left ventricular assist device analysis are beginning to distinguish changes associated with recovery from those of mechanical unloading alone and identify potential predictors and novel therapeutic targets capable of enhancing myocardial repair. SUMMARY: Significant progress has been made toward revealing molecular changes associated with myocardial recovery from heart failure. These studies also offer new insight into the pathogenesis of heart failure and point to novel therapeutic strategies.     

Calcium transport in erythrocytes of rats with spontaneous hypertension

In Quin-2-loaded erythrocytes of two genetically hypertensive rat strains (spontaneously hypertensive rats, SHR, and the Milan hypertensive strain, MHS) intracellular Ca2+ (Ca2+i) concentration and 45Ca influx rate were increased by 25-30 and 15-20% respectively, in comparison with normotensive controls (Wistar-Kyoto rats, WKY, and rats of the Milan normotensive strain, MNS). After 4 h incubation in the presence of 5 mmol/l sodium vanadate (Na3VO4) as an inhibitor of Ca-ATPase, 45Ca content of intact erythrocytes of SHR was twofold higher while erythrocyte count of stroke-prone SHR (SHRSP) was threefold higher than in WKY. This increase was observed in SHR during the pre-hypertensive stage. Under the same conditions, no difference was noted between MHS and MNS rats. The rate of 32P influx, as well as the concentration of exchangeable chloride, was studied. We failed to detect any significant differences in either parameter between hypertensive and normotensive rats, suggesting that altered cell membrane potential was not responsible for allied Ca fluxes. Erythrocyte shrinking, however, resulted in a two to threefold increase in the rate of 45Ca influx. Neither the rate of 45Ca influx nor Ca2+i were modified by the inhibitor of calmodulin-dependent reactions, R24571 (10 mumol/l). It is suggested that the higher rate of Ca2+ influx in Quin-2-loaded erythrocytes of SHR, as well as the increment in 45Ca content in intact erythrocytes treated with orthovanadate, is due to a change in membrane skeleton organization and cell shrinkage.     

Carvedilol decreases elevated oxidative stress in human failing myocardium

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of heart failure. However, direct evidence of oxidative stress generation in the human failing myocardium has not been obtained. Furthermore, the effect of carvedilol, a vasodilating beta-blocker with antioxidant activity, on oxidative stress in human failing hearts has not been assessed. This study was therefore designed to determine whether levels of lipid peroxides are elevated in myocardia of patients with dilated cardiomyopathy (DCM) and whether carvedilol reduces the lipid peroxidation level. Methods and Results- Endomyocardial biopsy samples obtained from 23 patients with DCM and 13 control subjects with normal cardiac function were studied immunohistochemically for the expression of 4-hydroxy-2-nonenal (HNE)-modified protein, which is a major lipid peroxidation product. Expression of HNE-modified protein was found in all myocardial biopsy samples from patients with DCM. Expression was distinct in the cytosol of cardiac myocytes. Myocardial HNE-modified protein levels in patients with DCM were significantly increased compared with the levels in control subjects (P<0.0001). Endomyocardial biopsy samples from 11 patients with DCM were examined before and after treatment (mean, 9+/-4 months) with carvedilol (5 to 30 mg/d; mean dosage, 22+/-8 mg/d). After treatment with carvedilol, myocardial HNE-modified protein levels decreased by 40% (P<0.005) along with amelioration of heart failure. CONCLUSIONS: Oxidative stress is elevated in myocardia of patients with heart failure. Administration of carvedilol resulted in a decrease in the oxidative stress level together with amelioration of cardiac function.     

The balance between pro-apoptotic and anti-apoptotic pathways in the failing myocardium

The purpose of this review is to highlight those circulating molecules, membrane receptors, and signaling pathways that initiate, potentiate, or conversely, inhibit apoptosis within cardiomyocytes. This review focuses on pathways directly related to the failing heart and discusses the limitations of current methodologies for assessing cardiomyocellular apoptosis. It is important to note that the adrenergic, reactive oxygen species, and proinflammatory cytokine signaling pathways are not the only pro-apoptotic pathways active in the myocardium, nor are IL-6-related cytokine, calcineurin, and IGF-1/P13K/Akt signaling pathways the only anti-apoptotic pathways active in the myocardium. However, the are among the best-characterized apoptosis-mediating pathways and therefore they may serve as foundation for future studies aimed at identifying novel apoptotic regulating pathways active in cardiomyocytes. Considering the short history of studying cardiomyocellular apoptosis, a tremendous body of knowledge has been collected. Understandably, much more work remains. Tomorrow’s studies must (1) continue to examine the signaling pathways mediating cardiomyocellular apoptosis by focusing on the links to the ubiquitous apoptosis effectors, (2) use the expanding body of knowledge to develop more specific inhibitors of apoptosis, and then (3) confirm the causal relationship of cardiomyocellular apoptosis and cardiac dysfunction in physiologic models of cardiac challenge.