Myocardial dysfunction in donor hearts. A possible etiology.

BACKGROUND: Potential cardiac donors show various degrees of myocardial dysfunction, and the most severely affected hearts are unsuitable for transplantation. The cause of this acute heart failure is poorly understood. We investigated whether alterations in calcium-handling proteins, beta-adrenoceptor density, or the inhibitory G protein Gialpha could account for this phenomenon in unused donor hearts (n=4 to 8). We compared these with end-stage failing hearts (n=14 to 16) and nonfailing hearts (n=3 to 12). METHODS AND RESULTS: Myocardial samples were obtained from unused donor hearts displaying ejection fractions <30%. Both trabeculae and isolated myocytes responded as poorly as those from the group of failing hearts to increasing stimulation frequency with regard to inotropic function in vitro. Immunodetectable abundance of sarcoplasmic reticulum calcium-ATPase and sodium calcium exchanger were greater (177%; P<0.01) and smaller (29%; P<0.01), respectively, in the unused donor hearts relative to the failing group, which suggests that alterations of these proteins are not a common cause of contractile dysfunction in the 2 groups. Myocytes from the unused donor group were desensitized to isoprenaline to a similar degree as those from the failing heart group. However, beta-adrenoceptor density was reduced in the failing (P<0.001) but not in the unused donor heart group (P=0.37) relative to the nonfailing heart group (n=5). Gialpha activity was increased in samples from unused donor and failing hearts relative to nonfailing hearts (P<0.05). CONCLUSIONS: Increased activity of the inhibitory G protein Gialpha is a significant contributory factor for impaired contractility in these acutely failing donor hearts.     

Quantitation and distribution of beta-tubulin in human cardiac myocytes

Increasing evidence suggests that derangements of cytoskeletal proteins contribute to alterations in intracellular signaling, myocyte function, and the coupling of myocytes to the extracellular matrix during cardiac hypertrophy and failure. Data from animal studies have shown an increased density of beta-tubulin protein in the right or left ventricle subjected to pressure overload, and have demonstrated that interfering with excess polymerization of beta-tubulin improves contractility. We tested the hypothesis that beta-tubulin is increased in human left ventricular hypertrophy and end-stage heart failure. Confocal microscopy of fluorescently labeled beta-tubulin protein revealed an increased density of the beta-tubulin network in cardiomyocytes from both hypertrophied and failing human hearts as compared to cells from nonfailing hearts. Western blot analysis on total heart homogenate showed no change in beta-tubulin when data were normalized to either actin or calsequestrin, although there was a significant increase in failing human hearts when data were normalized only for a constant amount of protein per heart. The mRNA for beta-tubulin was not changed in hypertrophied hearts, but was significantly decreased in failing human hearts. Thus, similar to animal models, we have shown that the density of the microtubular network within the cardiomyocyte is increased in end-stage failing human hearts. We have also shown for the first time that beta-tubulin density is increased in cells from hypertrophied human hearts. Although the functional implications of this finding in the human heart remain to be explored, data from animal studies suggest that increased beta-tubulin protein contributes to cardiac dysfunction.     

Increased myocardial NADPH oxidase activity in human heart failure

OBJECTIVES: This study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BACKGROUND: A phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis. An increase in myocardial oxidative stress due to excessive production of ROS may be involved in the pathophysiology of congestive heart failure. Recent studies have suggested an important role for myocardial NADPH oxidase in experimental models of cardiac disease. However, it is unknown whether NADPH oxidase is expressed in the human myocardium or if it has any role in human heart failure. METHODS: Myocardium of explanted nonfailing (n = 9) and end-stage failing (n = 13) hearts was studied for the expression of NADPH oxidase subunits and oxidase activity. RESULTS: The NADPH oxidase subunits p22(phox), gp91(phox), p67(phox), and p47(phox) were all expressed at messenger ribonucleic acid and protein level in cardiomyocytes of both nonfailing and failing hearts. NADPH oxidase activity was significantly increased in end-stage failing versus nonfailing myocardium (5.86 +/- 0.41 vs. 3.72 +/- 0.39 arbitrary units; p < 0.01). The overall level of oxidase subunit expression was unaltered in failing compared with nonfailing hearts. However, there was increased translocation of the regulatory subunit, p47(phox), to myocyte membranes in failing myocardium. CONCLUSIONS: This is the first report of the presence of NADPH oxidase in human myocardium. The increase in NADPH oxidase activity in the failing heart may be important in the pathophysiology of cardiac dysfunction by contributing to increased oxidative stress.     

Impaired angiotensin II-extracellular signal-regulated kinase signaling in failing human ventricular myocytes

Angiotensin II was reported to induce insulin-like growth factor-I and endothelin-1 gene expression and peptide release by ventricular cardiomyocytes. However, the progression from cardiac hypertrophy to failure in humans is characterized by a reduced myocyte expression of insulin-like growth factor-I and endothelin-1, notwithstanding the enhanced cardiac generation of angiotensin II. In the present study we investigated the functional status of the signaling pathways responsible for angiotensin II-induced endothelin-1 and insulin-like growth factor-I formation in human ventricular myocytes isolated from patients with dilated (n = 19) or ischemic (n = 14) cardiomyopathy and nonfailing donor hearts (n = 6).In human nonfailing ventricular myocytes, angiotensin II (100 nmol/l) induced insulin-like growth factor-I and endothelin-1 gene expression, and peptide release was mediated by extracellular signal-regulated kinase activation and inhibited by extracellular signal-regulated kinase antagonism (PD98059, 30 micromol/l), endothelin-1 formation being partially reduced also by c-Jun N-terminal kinase inhibition (SP600125, 10 micromol/l); insulin-like growth factor-I and endothelin-1 formations were unaffected by the inhibition of p38 mitogen-activated protein kinase (SB203580, 10 micromol/l) and Janus tyrosine kinase 2 (AG490, 10 micromol/l). In failing myocytes, angiotensin II failed to induce insulin-like growth factor-I and endothelin-1 formation; angiotensin II-induced extracellular signal-regulated kinase activation was significantly impaired (-88% vs. controls) although c-Jun NH2-terminal kinase activation was preserved. The impaired extracellular signal-regulated kinase phosphorylation in failing myocytes was associated with increased myocyte levels of mitogen-activated protein kinase phosphatases.Therefore, the altered growth factor production in failing myocytes is associated with a significant derangement in intracellular signaling.     

Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart

BACKGROUND: An increase in oxidative stress is suggested to be intimately involved in the pathogenesis of heart failure. However, gene expression of enzymes that metabolize reactive oxygen metabolites has not been investigated in the human heart. METHODS AND RESULTS: Myocardial tissue homogenates of the left ventricular wall from hearts in end-stage failure due to dilated (DCM) or ischemic (ICM) cardiomyopathy (n=12 each), as well as from nonfailing donor hearts (n=12), were analyzed for mRNA levels of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPX), and catalase by Northern blot analyses. Protein levels of MnSOD, CuZnSOD, and catalase were determined by Western blot or ELISA. MnSOD, CuZnSOD, and GPX mRNA levels were similar in all 3 groups. In contrast, catalase mRNA levels were found to be increased by 123+/-23% in DCM hearts and by 93+/-10% in ICM hearts (P<0.01 each) compared with control hearts. Likewise, catalase protein levels were found to be increased in failing hearts (DCM by 90+/-10%, ICM by 90+/-13%; P<0. 05 each) compared with control hearts. In addition, the observed upregulation of catalase mRNA and protein in failing hearts was attended by an increased catalase enzyme activity (DCM by 124+/-16%, ICM by 117+/-15%; P<0.01 each), whereas MnSOD, CuZnSOD, and GPX enzyme activity levels were unchanged in failing compared with nonfailing myocardium. CONCLUSIONS: Increased oxidative stress in human end-stage heart failure may result in a specific upregulation of catalase gene expression as a compensatory mechanism, whereas SOD and GPX gene expression remain unaffected.     

Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human heart.

Short duration exposure to cellular stresses have been shown to activate p38 mitogen-activated protein kinase (MAPK) in cultured rat ventricular cardiomyocytes and isolated perfused hearts; however, effects of chronic stress on p38 MAPK are not well understood. This study determined whether alterations in the p38 MAPK pathway occurred prior to end-stage human heart failure. The p38 MAPK alpha isoform was detectable in human cardiac tissue. However, carefully controlled analysis of protein and message in this study demonstrated an absence of the p38 MAPK beta -isoform. Low levels of message for the non-SB203580 sensitive p38 MAPK gamma and delta isoforms were also detected in both normal and failing human myocardium. Ischemic and idiopathic end-stage failing human hearts were compared to non-failing hearts for both p38 alpha MAPK protein level and total p38 MAPK activity. Western blotting techniques demonstrated no significant changes in total p38 alpha MAPK content. However, approximately 75% decreases in active/phosphorylated p38 MAPK (P<0.005) were observed in both ischemic and idiopathic failing hearts compared to non-failing hearts. In-gel kinase assays confirmed that activated p38 MAPK, detected by Western blotting, phosphorylated its potential downstream targets. When compared to non-failing hearts, approximately 46% decreases in p38 MAPK phosphorylation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) were observed in ischemic and idiopathic failing hearts (P=0.03 and P=0.04 respectively). Active p38 MAPK was localized to sarcomeric structures in the cytosol of myocytes by confocal immunofluorescence microscopy. The correlation between decreased MAPKAPK-2 phosphorylation and loss of active p38 MAPK in failing human myocytes suggests that decreases in the activation of p38 MAPK alpha, the predominant cardiac isoform, occur prior to end-stage heart failure.     

Myosin heavy chain isoform expression in the failing and nonfailing human heart

In the heart, the relative proportions of the 2 forms of the motor protein myosin heavy chain (MyHC) have been shown to be affected by a wide variety of pathological and physiological stimuli. Hearts that express the faster MyHC motor protein, alpha, produce more power than those expressing the slower MyHC motor protein, beta, leading to the hypothesis that MyHC isoforms play a major role in the determination of cardiac contractility. We showed previously that a significant amount of alphaMyHC mRNA is expressed in nonfailing human ventricular myocardium and that alphaMyHC mRNA expression is decreased 15-fold in end-stage failing left ventricles. In the present study, we determined the MyHC protein isoform content of human heart samples of known MyHC mRNA composition. We demonstrate that alphaMyHC protein was easily detectable in 12 nonfailing hearts. alphaMyHC protein represented 7.2+/-3.2% of total MyHC protein (compared with approximately 35% of the MyHC mRNA), suggesting that translational regulation may be operative; in contrast, there was effectively no detectable alphaMyHC protein in the left ventricles of 10 end-stage failing human hearts.     

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure

OBJECTIVES: Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS: We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS: Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION: The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.     

Force/shortening-frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts.

BACKGROUND: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. METHODS AND RESULTS: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5-2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). CONCLUSIONS: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts.

OBJECTIVES: A hallmark of human heart failure is prolonged myocardial relaxation. Although the intrinsic mechanism of phospholamban coupling to the Ca(2+)-ATPase is unaltered in normal and failed human hearts, it remains possible that regulation of phospholamban phosphorylation by cAMP-dependent mechanisms or other second messenger pathways could be perturbed, which may account partially for the observed dysfunctions of the sarcoplasmic reticulum (SR) associated with this disease. METHODS: cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were characterized initially by DEAE-Sepharose chromatography in hearts from patients with end-stage dilated cardiomyopathy. We measured the activity of PKA and CaM kinase in left ventricular tissue of failing (idiopathic dilated cardiomyopathy; ischemic heart disease) and nonfailing human hearts. RESULTS: Basal PKA activity was not changed between failing and nonfailing hearts. One major peak of CaM kinase activity was detected by DEAE-Sepharose chromatography. CaM kinase activity was increased almost 3-fold in idiopathic dilated cardiomyopathy. In addition, hemodynamical data (left ventricular ejection fraction, cardiac index) from patients suffering from IDC positively correlate with CaM kinase activity. CONCLUSIONS: Increased CaM kinase activity in hearts from patients with dilated cardiomyopathy could play a role in the abnormal Ca2+ handling of the SR and heart muscle cell.     

Increased messenger RNA level of the inhibitory G protein alpha subunit Gi alpha-2 in human end-stage heart failure.

In human heart failure the positive inotropic and cAMP-elevating effects of both beta-adrenoceptor agonists and phosphodiesterase inhibitors are diminished. This has been explained at least in part by an increase in the inhibitory signal-transducing G protein (Gi) and unchanged stimulatory G protein (Gs). In the present study we determined the mRNA expression pattern of the alpha subunits of Gi-1, Gi-2, Gi-3, and Gs in myocardial tissue samples of patients undergoing heart transplantation. Northern blot analysis of total RNA extracted from left ventricles with 32P-labeled cDNAs demonstrated expression of Gi alpha-2, Gi alpha-3, and Gs alpha mRNA. In contrast, Gi alpha-1 mRNA was not detectable. To investigate whether the increased ratio of Gi/Gs might be due to altered gene expression, we compared mRNA levels of Gi alpha-2, Gi alpha-3, and Gs alpha in left ventricular myocardium from failing hearts with idiopathic dilated cardiomyopathy (n = 8) and ischemic cardiomyopathy (n = 6) and from nonfailing hearts from transplant donors (n = 8). Compared with nonfailing control hearts, the Gi alpha-2 mRNA was increased by 75 +/- 26% (p less than 0.05) in idiopathic dilated cardiomyopathy hearts and 90 +/- 26% (p less than 0.05) in ischemic cardiomyopathy hearts. Gi alpha-3 and Gs alpha mRNA levels were similar in the three groups. The results suggest that as in other mammalian species, Gi alpha-2 and Gi alpha-3 mRNA are the predominant Gi alpha mRNA subtypes in human ventricular myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased expression of constitutive nitric oxide synthase III,but not inducible nitric oxide synthase II,in human heart failure

Objectives. The purpose of the present study was to examine the expression of the endothelial-type nitric oxide synthase (NOS III) and the inducible-type NOS (NOS II) in human myocardium and their regulation in heart failure from patients with different etiologies.Background. In heart failure, plasma levels of nitrates were found to be elevated. However, data on myocardial NOS expression in heart failure are conflicting.Methods. Using RNase protection analysis and Western blotting, the expression of NOS III and NOS II was investigated in ventricular myocardium from nonfailing (NF) hearts (n = 5) and from failing hearts of patients with idiopathic dilated cardiomyopathy (dCMP, n = 14), ischemic cardiomyopathy (iCMP, n = 9) or postmyocarditis cardiomyopathy (mCMP, n = 7). Furthermore, immunohistochemical studies were performed to localize NOS III and NOS II within the ventricular myocardium.Results. In failing human hearts, NOS III mRNA levels were increased to 180% in dCMP, 200% in iCMP and to 210% in mCMP as compared to NF hearts. Similarly, in Western blots (using constitutively expressed beta-tubulin as a reference) NOS III protein expression was increased about twofold in failing compared to NF hearts. Immunohistochemical studies with a selective antibody to NOS III showed no obvious differences in the staining of the endothelium of cardiac blood vessels from NF and failing human hearts. However, NOS III-immunoreactivity in cardiomyocytes was significantly more intense in failing compared to NF hearts. Low expression of NOS II mRNA was detected in only 2 of 30 failing human hearts and was not found in NF hearts. Inducible-type NOS protein was undetectable in either group.Conclusions. We conclude that the increased NOS III expression in the ventricular myocardium of failing human hearts may contribute to the contractile dysfunction observed in heart failure and/or may play a role in morphologic alterations such as hypertrophy and apoptosis of cardiomyocytes.     

Force/shortening[ndash ]frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts

Background: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. Methods and Results: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; N = 11) and nonfailing (donor hearts; N = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5[ndash ]2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). Conclusions: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.