Myocyte Cell Loss Research Articles

Abstract 545: Generation of Oxidized Phosphatidylcholines During Hypoxia-Reoxygenation Triggers Cell Death of Postnatal Cardiac Myocytes

Reperfusion of ischemic heart tissue leads to a rapid burst of reactive oxygen species which initiates a cascade of events that cumulate in cell death. The most susceptible biomolecules to oxidative injury are membrane associated phospholipids. Oxidized phospholipids including oxidized phosphatidylcholines (OxPC), have shown to be potent activators of the intrinsic apoptotic signaling cascade. We hypothesize that the production of OxPC within hypoxic myocytes mediates damage from hypoxia-reoxygenation (H-R) by triggering inappropriate cardiac myocytes death. Hence, we determined whether, bioactive OxPC are produced in cardiomyocytes during H-R, and whether OxPC species induces apoptotic response resulting in cell death of cardiomyocytes. Post-natal rat cardiomyocytes were isolated from 2 d old rat pups and submitted to primary culture in serum-free medium. Cardiac myocytes were subjected to hypoxia (H) for 1 hour in a specialized gas-tight chamber under continuous nitrogen atmosphere followed by 1 hour reoxygenation (R). Lipidomic analysis of cardiac myocytes following H-R was determined by high performance liquid chromatography linked to tandem mass spectrometry was used to quantitate 82 distinct OxPC species from cell lipid extracts. Cell viability of cardiomyocytes was assessed in the absence and presence of known OxPC molecules (POVPC, PONPC, PGPC, PAzPC) and control non-oxidized PSPC. Lipidomic analysis of OxPC species showed increased generation of fragmented OxPC after H-R (10.20±0.67μg/mL vs1.95±0.24μg/mL, p<0.005) when compared to control cells. Cells exposed to POVPC and PONPC showed marked dose dependent increase in cell death compared to equal molar concentration of PSPC. There was also marked increase in mitochondrial permeability upon exposure to fragmented OxPC molecules. To our knowledge this is the first demonstration that OxPC are generated within post-natal cardiomyocytes during H-R and potent inducers of cell death of cardiac myocyte during H-R. Interventions designed to modulate the synthesis of cytotoxic OxPC during H-R may prove beneficial in suppressing aberrant cardiac myocyte cell death and loss of ventricular function in patients with ischemic heart disease or following myocardial infarction.

The Myocardial Unfolded Protein Response during Ischemic Cardiovascular Disease.

Heart failure is a progressive and disabling disease. The incidence of heart failure is also on the rise, particularly in the elderly of industrialized societies. This is in part due to an increased ageing population, whom initially benefits from improved, and life-extending cardiovascular therapy, yet ultimately succumb to myocardial failure. A major cause of heart failure is ischemia secondary to the sequence of events that is dyslipidemia, atherosclerosis, and myocardial infarction. In the case of heart failure postmyocardial infarction, ischemia can lead to myocardial cell death by both necrosis and apoptosis. The extent of myocyte death postinfarction is associated with adverse cardiac remodeling that can contribute to progressive heart chamber dilation, ventricular wall thinning, and the onset of loss of cardiac function. In cardiomyocytes, recent studies indicate that myocardial ischemic injury activates the unfolded protein stress response (UPR) and this is associated with increased apoptosis. This paper focuses on the intersection of ischemia, the UPR, and cell death in cardiomyocytes. Targeting of the myocardial UPR may prove to be a viable target for the prevention of myocyte cell loss and the progression of heart failure due to ischemic injury.

Modulation of Programmed Forms of Cell Death by Intracoronary Levosimendan During Regional Myocardial Ischemia in Anesthetized Pigs

Powerful mediators of programmed cell death, such as apoptosis and autophagy, can contribute to myocyte cell loss during pathological cardiac conditions. Levosimendan has been shown to exert beneficial hemodynamic effects in presence of global myocardial ischemia and heart failure through vasodilatation and increase of cardiac contractility. Recently, the intracoronary administration of a bolus levosimendan was found to exert favourable cardiac anti-stunning effects without lowering arterial pressure, which limits the use of levosimendan mainly in coronary artery disease. Here we tested whether the intracoronary administration of levosimendan can beneficially modulate programmed cell death in acute regional myocardial ischemia. Acute regional myocardial ischemia was induced in 20 anaesthetized pigs and intracoronary levosimendan 15 min bolus administration was started 4 h afterwards. The effects of levosimendan on coronary blood flow and cardiac function were evaluated and myocardial biopsies were examined for criteria of autophagy and apoptosis. The administration of levosimendan caused a significant increase of coronary blood flow (p < 0.05) in absence of changes in cardiac function. Moreover, levosimendan prevented the down-regulation of the anti-apoptotic gene, Bcl-2, and the up-regulation of the apoptotic markers Bax and cytochrome c, which resulted in a reduced expression of TUNEL fragmented nuclei (p < 0.05). Furthermore, levosimendan maintained Beclin 1 at 4 h and potentiated LC3 II expression, these results being consistent with autophagy activation. Such effects of intracoronary levosimendan bolus administration during regional myocardial ischemia indicate the occurrence of cardio-protection by modulation of programmed form of cell death.

Pathophysiologie der chronischen Herzinsuffizienz

Heart failure is a progressive and often fatal disease process. In general, the pathophysiologic mechanisms responsible for progressive myocyte dysfunction and cell loss, cardiac remodeling and arrhythmias involve signaling mechanisms that alter myocardial gene expression. These changes in gene expression are complex and involve contractile proteins, ion channels, Ca(++) handling, apoptosis, cell metabolism, the extracellular matrix, signal transduction pathways and growth factors. In the failing heart, several changes occur in cardiac adrenergic receptor-signal transduction pathways. The most striking of these changes occur in beta-adrenergic receptors, and of the changes in beta-adrenergic receptors beta1-receptor down-regulation is the most prominent. Other changes include uncoupling of beta2-adrenergic receptors and increased activity of the inhibitory G-protein. Most of these changes appear to be related to increased activity of the adrenergic nervous system, i.e. increased exposure to norepinephrine. Antagonists of the adrenergic nervous system may improve left ventricular function and outcome in patients with heart failure. This fact supports the notion that activation of these neurohormonal systems exerts a net long-term detrimental effect on the natural history of chronic heart failure and that myocardial adrenergic desensitization phenomena are at least partially maladaptive in the setting of left ventricular dysfunction. In addition to functional alterations structural remodeling plays a major role in the progression of various heart diseases to congestive heart failure. Major contributors to this remodeling process in the heart include alterations in myocyte shape, myocyte number and extracellular matrix. However, it is unclear as to which of these changes is most critical in the development of congestive heart failure, and this may vary by etiology.

Clinical Implications of Apoptosis in Ischemic Myocardium

Apoptosis, a genetically programmed form of cell death, contributes to myocyte cell loss in a variety of cardiac pathologies, including cardiac failure and those related to ischemia/reperfusion injury. The apoptotic program is complex, involving both pro- and anti-apoptotic proteins, and apoptosis occurs when the equilibrium between these opposing factors is perturbed. Some of these factors are intrinsic to the apoptotic pathway, such as the pro- and anti-apoptotic members of the Bcl2 family. Other, extrinsic, cellular factors can also modify the outcome of the response to an apoptotic stimulus. In this review, we have focused on some of these extrinsic factors, such as STAT-1 as a pro-apoptotic agent and the urocortins and Bag-1 as anti-apoptotic factors, since these may be potential therapeutic targets. In addition, we discuss the profound cytoprotective effects of the antibiotic, minocycline.

Cellular events linked to cardiac remodeling in heart failure: targets for pharmacologic intervention.

Over the past decade, there has been a paradigm shift in the understanding of heart failure pathophysiology. Heart failure is no longer conceptualized as a hemodynamic disorder resulting from changes in renal and hormonal function. Rather, the syndrome of heart failure is more complex and is characterized by abnormal myocyte growth, proliferation of cells in the extracellular matrix, and myocyte cell loss (apoptosis)--all of which culminate in significant structural remodeling of the heart and loss of ventricular function. The loss of ventricle function is preceded by an initiating event such as myocardial infarction, which leads to changes in cell function, activation of specific neurohormones and peptides, which in turn are linked to the remodeling of the ventricle, and progression of heart failure. This article discusses how changes in myocyte and nonmyocyte structure may contribute to the progression of heart failure. Insight into these mechanisms will provide a better understanding of newer pharmacologic approaches in the treatment of heart failure.

Apoptotic Cell Death in Arrhythmogenic Right Ventricular Cardiomyopathy. A Comparative Study with Idiopathic Sustained Ventricular Tachycardia.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a specific heart muscle disease of unknown etiology characterized by fatty and fibrofatty replacement of the right ventricular myocardium. It often manifests life-threatened ventricular arrhythmias. Previous studies have hypothesized that myocyte apoptosis contributes to the myocyte cell loss and fatty change in ARVC and may be induced by recurrent ventricular tachycardia (VT). We examined whether these progressive pathological changes result from apoptotic cell death in both autopsied and biopsied right ventricular myocardium from 35 patients with ARVC by using in situ terminal deoxynucleotidyl transferase assay (TUNEL) and agarose gel electrophoresis. We also studied the biopsied myocardium from 30 patients with idiopathic sustained VT whose origin was the outflow tract of the right ventricle. TUNEL-positive cells indicating DNA fragments were observed in some cardiomyocytes and fibroblasts in ARVC, but the numbers of TUNEL-positive myocytes were very low in idiopathic VT. DNA laddering was confirmed in two autopsied cases in ARVC, but not in a non-cardiac case who died. These results suggest that at least some cardiomyocytes and fibroblasts are subjected to apoptosis in ARVC, leading to the loss of myocardium with characteristic pathological changes and subsequently progressive cardiomyopathy. Furthermore, the apoptotic process may not result from myocardial ischemia due to repetitive VT.

Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis.

The extent to which apoptosis contributes to myocyte cell loss during acute carditic viral infection is unknown. To assess whether apoptosis occurs in acute viral myocarditis, and how it is modulated, we studied mice inoculated with coxsackievirus B3 (CVB3). Five CD1 and C3H.HeJ (C3H) mice/group were sacrificed as saline vehicle-injected controls, and at 1, 2, and 3 weeks post-inoculation (p.i.) with 5 x 10(6) pfu CVB3. Histopathological status and terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays quantified inflammation, necrosis and apoptosis in myocardium. Apoptosis-related protein immunoreactivity defined presence and location of Bax, Fas, Fas Ligand (FasL), Bcl-2, interleukin-1 beta converting enzyme (ICE), inducible nitric oxide synthase (iNOS) and the proto-oncogene p53. Both strains exhibited significant histopathology at all time points. Saline-injected control animals showed no signs of inflammation and no significant difference in apoptosis-related protein immunoreactivity was observed between strains. Myocardial TUNEL-positive cells were exceedingly rare though apoptosis was present in thymic medulla and spleen follicles. Pro-apoptotic proteins Bax, Fas, and FasL were present in all groups though no clear correlation with histopathology was apparent. By contrast, the anti-apoptotic protein Bcl-2 showed mild immunoreactivity in controls, which increased following infection and correlated well with histopathological scores in both strains. Myocardial iNOS immunoreactivity displayed a similar though weaker staining pattern to Bcl-2 over the 3 week study period in both strains. Neither ICE nor p53 immunoreactivity could be demonstrated in myocardium. Thus, despite marked inflammatory activity, myocyte apoptosis is rare in acute CVB3 myocarditis in CD1 and C3H.HeJ mice.

Myocyte Cell Loss in Ischemic Cardiomyopathy: Role of Apoptosis

Myocyte Cell Loss in Ischemic Cardiomyopathy: Role of Apoptosis

P53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.

Electrophysiological Remodeling of the Heart Owing to Rate

Although damage to the heart from inflammation and infarction are well-established stimuli that trigger major cardiac remodeling, it has been known since 1954 that a persistent supraventricular tachycardia also can alter cardiac structure and function.1 This clinical observation was exploited 8 years later when it was demonstrated that rapid stimulation of the normal heart could produce reversible congestive heart failure.2 Since then, rapid pacing has been used frequently to create an investigational animal model of heart failure. The effects of prolonged pacing-induced tachycardia, in the range of 250 bpm for 3 or 4 weeks, has been studied in depth in several animal species and has been shown to cause extensive myocardial remodeling. Biventricular enlargement with reduced wall thickness and no increase in muscle mass, reduced cross-sectional myocyte area and increased length, disarray of myofibril alignment and cytoarchitecture, increases in contractile protein degradation, and destruction of the supporting myocardial collagen matrix represent some of the structural changes contributing to reduced ventricular function.3 Diminished responsiveness to β-adrenoceptor stimulation caused by a reduction in β-receptor density and a variety of alterations in the adrenergic signaling processes also occur.4 Replacement fibrosis, loss of cells, and reactive hypertrophy of the remaining cells have been noted recently.5 mRNA levels for specific cytoskeletal proteins increase, along with alterations in cytoskeletal architecture6 and abnormalities in phospholamban.7 In pigs subjected to 3 weeks of a tachycardia-induced cardiomyopathy, myocyte action potential showed reduced membrane resting potential, reduced amplitude and upstroke velocity, and prolonged duration at 90% repolarization. Peak L-type Ca2+ current density was reduced because of an absolute reduction in L-type Ca2+ current flow into the myocyte possibly caused by reduced open probability of the channel.8 However, in dogs that had rapid ventricular pacing for 3 to 4 weeks, action …

Gender differences and aging: Effects on the human heart

This study investigated the changes in myocyte size and number in the left and right ventricles that occur with aging in the female and male heart. Differences in life span between women and men may be related to a better preservation of myocardial structure in the female heart with aging. On this basis, the hypothesis was advanced that the aging process has a different impact on the integrity of the myocardium in the two genders. Morphometric methodologies were applied to analyze the changes in number and size of ventricular myocytes in the hearts of 53 women and 53 men. The changes in mononucleated and binucleated myocytes with age were determined in enzymatically dissociated cells. The age interval examined varied from 17 to 95 years. Aging was associated with a preservation of ventricular myocardial mass, aggregate number of mononucleated and binucleated myocytes, average cell diameter and volume in the female heart. In contrast, nearly 1 g/year of myocardium was lost in the male heart, and this phenomenon accounted for the loss of approximately 64 million cells. This detrimental effect involved the left and right sides of the heart. In the remaining cells, myocyte cell volume increased at a rate of 158 microns3/year in the left and 167 microns3/year in the right ventricle. Aging does not lead to myocyte cell loss and myocyte cellular reactive hypertrophy in women, indicating that gender differences may play a significant role in the detrimental effects of the aging process on the heart.

Ischemic cardiomyopathy: myocyte cell loss, myocyte cellular hypertrophy, and myocyte cellular hyperplasia.

Ischemic cardiomyopathy: myocyte cell loss, myocyte cellular hypertrophy, and myocyte cellular hyperplasia.

The cellular basis of dilated cardiomyopathy in humans

The present investigation was designed to evaluate whether end-stage cardiac failure in patients affected by dilated cardiomyopathy (DC) was dependent upon extensive myocyte cell death with reduction in muscle mass or was the consequence of collagen accumulation in the myocardium independently from myocyte cell loss. In addition, the mechanisms of ventricular dilation were analysed in order to determine whether the changes in cardiac anatomy were important variables in the development of intractable congestive heart failure. DC is characterized by chamber dilation, myocardial scarring and myocyte hypertrophy in the absence of significant coronary atherosclerosis. However, the relative contribution of each of these factors to the remodeling of the ventricle is currently unknown. Moreover, no information is available concerning the potential etiology of collagen deposition in the myocardium and the changes in number and size of ventricular myocytes with this disease. Morphometric methodologies were applied to the analysis of 10 DC hearts obtained from patients undergoing cardiac transplantation. An identical number of control hearts was collected from individuals who died from causes other than cardiovascular diseases. DC produced a 2.2-fold and 4.2-fold increase in left ventricular weight and chamber volume resulting in a 48% reduction in mass-to-volume ratio. In the right ventricle, tissue weight and chamber size were both nearly doubled. Left ventricular dilation was the result of a 59% lengthening of myocytes and a 20% increase in the transverse circumference due to slippage of myocytes within the wall. Myocardial scarring represented by segmental, replacement and interstitial fibrosis occupied approximately 20% of each ventricle, and was indicative of extensive myocyte cell loss. However, myocyte number was not reduced and average cell volume increased 2-fold in both ventricles. In conclusion, reactive growth processes in myocytes and architectural rearrangement of the muscle compartment of the myocardium appear to be the major determinants of ventricular remodeling and the occurrence of cardiac failure in DC.

Nonischemic myocardial damage induced by nonocclusive constriction of coronary artery in rats

To determine whether reduction in coronary vessel diameter leads to alterations in cardiac function, coronary perfusion, and tissue integrity, the left coronary artery of rats was narrowed and ventricular hemodynamics measured at 3 and 5 days after surgery. Coronary artery narrowing averaged 62% and end-diastolic pressure was increased, whereas peak systolic pressure, positive change in pressure over time, stroke volume, and total peripheral resistance were decreased. However, this impairment of function was accompanied by a preservation of resting coronary blood flow (CBF), although a 43% decrease in maximal CBF was detected. Foci of reparative fibrosis and myocytolytic necrosis were found primarily in the endomyocardium and midmyocardium. These lesions were temporally distinct, corresponding to 5 days and 12- to 24-h-old forms of myocardial damage, respectively. The changes in maximal CBF correlated with the degree of stenosis, whereas the volume fraction, average cross-sectional area, and number of foci of reparative fibrosis lesions per unit area of myocardium correlated exclusively with end-diastolic pressure. In conclusion, reductions in luminal diameter of a major coronary artery not affecting resting coronary perfusion have a profound detrimental impact on cardiac performance and initiate immediate myocyte cell loss that is ongoing. Thus tissue and cellular damage may not be ischemic in nature but rather mediated by other mechanisms such as unbearable mechanical stress.

Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart.

To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 4, 12, 20, and 29 months of age. Mean arterial pressure, left ventricular pressure and its first derivative (dP/dt), and heart rate were comparable in rat groups up to 20 months. During the interval from 20 to 29 months, elevated left ventricular end-diastolic pressure and decreased dP/dt indicated that a significant impairment of ventricular function occurred with senescence. In the period between 4 and 12 months, a reduction of nearly 19% in the total number of myocytes was measured in both ventricles. In the subsequent ages, similar decreases in myocyte cell number were found in the left ventricle, whereas in the right ventricle, the initial loss was fully reversed by 20 months. Moreover, from 20 to 29 months, a 59% increase in the aggregate number of myocytes occurred in the right ventricular myocardium. In the left ventricle, a 3% increment was also seen, but this small change was not statistically significant. These estimations of myocyte cellular hyperplasia, however, were complicated by the fact that cell loss continued to take place with age. The volume fraction of collagen in the tissue, in fact, progressively increased from 8% and 7% at 4 months to 16% and 22% at 29 months in the left and right ventricles, respectively. In conclusion, myocyte cellular hyperplasia tends to regenerate the ventricular mass being lost with age in the adult mammalian rat heart.

Myocyte cell loss and myocyte hypertrophy in the aging rat heart

To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 3, 10 to 12 and 19 to 21 months of age. Systemic arterial pressure, left ventricular pressure and its first derivative (dP/dt) and heart rate were comparable in the three animal groups. In the interval between 3 and 10 to 12 months, mean myocyte cell volume per nucleus increased 53 and 26% in the left and the right ventricle, respectively. The total number of myocyte nuclei remained constant in either ventricle. In the following period, between 10 to 12 and 19 to 21 months, a 39% further cellular hypertrophy on the left side of the heart was found in association with an 18% loss of cells in the ventricle. Cell loss was accompanied by discrete areas of interstitial and replacement fibrosis in the subendocardium. In contrast, no myocardial damage was observed in the right ventricle, and the measured 35% additional enlargement of myocytes occurred without a change in cell number. Thus, the aging left ventricle is composed of a smaller number of hypertrophied cells. Cellular hypertrophy may explain the unaltered cardiac function of the aged myocardium.

Myocardial DNA synthesis in experimental cardiac hypertrophy.

Myocardial DNA synthesis in experimental cardiac hypertrophy.