Combining SERCA2a activation and Na-K ATPase inhibition: a promising new approach to managing acute heart failure syndromes with low cardiac output.

Heart failure (HF) patients are a medically complex and heterogeneous population with multiple cardiac and non-cardiac comorbidities. Although there are a multitude of etiologic substrates and initiating and amplifying mechanisms contributing to disease progression, these pathophysiologic processes ultimately all lead to impaired myocardial function. The myocardium must both pump oxygenated, nutrient-rich blood throughout the body (systolic function) and receive deoxygenated, nutrient-poor blood returning from the periphery (diastolic function). At the molecular level, it is well-established that Ca2+ plays a central role in excitation-contracting coupling with action potentials stimulating the opening of L-type Ca2+ in the plasma membrane and ryanodine receptor 2 (RyR2) in the sarcoplasmic reticulum (SR) membrane during systole and the Na-Ca2+ exchanger and SERCA2a returning Ca2+ to the extracellular space and SR, respectively, during diastole. However, there is increasing recognition that impaired Ca2+ cycling may contribute to myocardial dysfunction. Preclinical studies and clinical trials indicate that combining SERCA2a activation and Na-K ATPase inhibition may increase contractility (inotropy) and facilitate active relaxation (lusitropy), improving both systolic and diastolic functions. Istaroxime, a novel luso-inotrope that activates SERCA2a and inhibits the Na-K ATPase, is currently in phase II clinical development and has been shown to improve systolic and diastolic functions and central hemodynamics, increase systolic but not diastolic blood pressure, and decrease substantially heart rate. Irrespective of its clinical utility, the development of istaroxime has evolved our understanding of the clinical importance of inhibiting the Na-K ATPase in order to obtain a clinically significant effect from SERCA2a activation in the setting of myocardial failure.

Bosentan enhances viral load via endothelin-1 receptor type-A-mediated p38 mitogen-activated protein kinase activation while improving cardiac function during coxsackievirus-induced myocarditis.

Reduced cardiac output is one of the consequences of myocarditis. Bosentan, an endothelin-1 receptor (ET1R) antagonist, could be useful to reduce cardiac afterload, preserving cardiac output. In this study, we investigated the potential therapeutic use of bosentan in an animal model of viral myocarditis. Using a mouse model of coxsackievirus B3 (CVB3)-induced myocarditis, we demonstrated preserved ejection fraction (EF) and fractional shortening (FS) by treatment with bosentan (68+/-5.8% EF and 40+/-3.7% FS for treated versus 48+/-2.2% EF and 25+/-2.6% FS for controls; P=0.028). However, bosentan enhanced cardiac viral load (10.4+/-6.7% in the bosentan group versus 5.0+/-5.5% in control group; P=0.02), likely through enhancement of p38 mitogen-activated protein kinase (MAPK) phosphorylation (0.77+/-0.40% ATF2 activation in the bosentan group versus 0.03+/-0.02% in controls; P=0.0002), mediated by endothelin receptor type-A. We further demonstrate that a water soluble inhibitor of p38 MAPK, SB203580 HCl, is a potent inhibitor of virus replication in the heart (0.28% antisense viral genome stained area for 3 mg/kg dose versus 2.9% stained area for controls; P=0.01), attenuates CVB3-induced myocardial damage (blinded cardiac histopathologic scores of 1.8+/-1.6 and 2.05+/-1.2 for the 3 mg/kg and 10 mg/kg doses, respectively, versus 3.25+/-1.2 for the controls), and preserves cardiac function (69+/-3.5% EF for 3 mg/kg dose and 71+/-6.7% EF for 10 mg/kg dose versus 60+/-1.5% EF control; P=0.038 and P=0.045, as compared to control, respectively). Bosentan, a prescribed vasodilator, improves cardiac function but enhances viral load and myocarditis severity through ETRA mediated p38 MAPK activation; p38 MAPK is a desirable antiviral target. Caution must be exercised during treatment of suspected infectious myocarditis with supportive vasoactive remedies.

Distribution and correlates of lipoprotein-associated phospholipase A2 in an elderly cohort: the Cardiovascular Health Study.

OBJECTIVES: To determine whether high levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) are associated with prevalent cardiovascular disease (CVD) and to evaluate factors most influencing Lp-PLA(2) levels in a community-based cohort of older adults. DESIGN: Cross-sectional. SETTING: The Cardiovascular Health Study (CHS), a population-based cohort study of men and women aged 65 and older. PARTICIPANTS: Five thousand five hundred thirty-one CHS participants. MEASUREMENTS: Levels of Lp-PLA(2) activity were determined using stored blood samples from the baseline examination. RESULTS: Mean Lp-PLA(2) was higher in participants with electrocardiographically determined ventricular conduction defect and major Q-wave abnormality and was positively correlated with left ventricular (LV) mass. It was high in those with echocardiographically determined abnormal LV ejection fraction, which persisted after adjustment. Mean Lp-PLA(2) was also higher in participants with mild renal insufficiency and kidney disease. After multivariable adjustment, there was a modest but significant 27% greater risk of prevalent CHF per standard deviation increment of Lp-PLA(2) and a modest but significant 12% greater risk of prevalent myocardial infarction. Lp-PLA(2) was weakly but mainly most strongly correlated with cholesterol and lipoproteins, but those correlations were not especially strong. Lp-PLA(2) was weakly positively correlated with soluble intercellular adhesion molecule-1 but not interleukin-6. In total, all factors considered could explain only 29% of Lp-PLA(2) activity. CONCLUSION: Novel findings in the study are the associations, in those aged 65 and older, between Lp-PLA(2) activity and LV dysfunction, CHF, and renal disease. CVD risk factors only minimally explain levels of Lp-PLA(2).

A cardiac myosin light chain kinase regulates sarcomere assembly in the vertebrate heart.

Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v), which is involved in the development of human cardiomyopathy, is an important structural protein that affects physiologic cardiac sarcomere formation and heart development. Integrated cDNA expression analysis of failing human myocardia uncovered a novel protein kinase, cardiac-specific myosin light chain kinase (cardiac-MLCK), which acts on MLC2v. Expression levels of cardiac-MLCK were well correlated with the pulmonary arterial pressure of patients with heart failure. In cultured cardiomyocytes, knockdown of cardiac-MLCK by specific siRNAs decreased MLC2v phosphorylation and impaired epinephrine-induced activation of sarcomere reassembly. To further clarify the physiologic roles of cardiac-MLCK in vivo, we cloned the zebrafish ortholog z-cardiac-MLCK. Knockdown of z-cardiac-MLCK expression using morpholino antisense oligonucleotides resulted in dilated cardiac ventricles and immature sarcomere structures. These results suggest a significant role for cardiac-MLCK in cardiogenesis.

Matrix metalloprotease activity is enhanced in the compensated but not in the decompensated phase of pressure overload hypertrophy.

BACKGROUND: During the transition of pressure overload hypertrophy (POH) to heart failure (HF) there is intense interstitial cardiac remodeling, characterized by a complex balance between collagen deposition and degradation by matrix metalloproteases (MMPs). This study was aimed at investigating the process of cardiac remodeling during the different phases of the transition of POH to HF. METHODS: Guinea pigs underwent thoracic descending aortic banding or sham operation. Twelve weeks after surgery, left-ventricular (LV) end-diastolic internal dimension and ventricular systolic pressure were measured by combined M-mode echocardiography and micromanometer cathetherization. The MMP activity, tissue-specific MMP inhibitors (TIMPs), and collagen fraction were evaluated in LV tissue samples by zymography, ELISA, and computer-aided analysis, respectively. RESULTS: Banded animals were divided by lung weight values into either compensated left-ventricular hypertrophy (LVH) or HF groups, as compared with sham-operated controls. All HF animals exhibited a restrictive pattern of Doppler transmitral inflow, indicative of diastolic dysfunction, and developed lung congestion. Compensated LVH was associated with increased MMP-2 activity, which was blunted after transition to HF, at a time when TIMP-2 levels and collagen deposition were increased. CONCLUSIONS: The cardiac remodeling process that accompanies the development of POH is a phase-dependent process associated with progressive deterioration of cardiac function.

Beta1 integrins modulate beta-adrenergic receptor-stimulated cardiac myocyte apoptosis and myocardial remodeling.

Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that beta1 integrins play a protective role in chronic beta-adrenergic receptor-stimulated cardiac myocyte apoptosis and heart failure. L-isoproterenol (iso; 400 microg/kg per hour) was infused in a group of wild-type (WT) and beta1 integrin heterozygous knockout (hKO) mice. Left ventricular structural and functional remodeling was studied at 7 and 28 days of iso-infusion. Western blot analysis demonstrated reduced beta1 integrin levels in the myocardium of hKO-sham. Iso-infusion increased heart weight:body weight ratios in both groups. However, the increase was significantly higher in WT-iso. M-mode echocardiography indicated increased left ventricular end-diastolic diameter, percentage of fractional shortening, and ejection fraction in the WT-iso group. The percentage of fractional shortening and ejection fraction were significantly lower in hKO-iso versus hKO-sham and WT-iso. Peak left ventricular developed pressure and left ventricular end-diastolic pressure measured using Langendorff-perfusion analyses were significantly higher in the WT-iso group (P<0.05 versus WT-sham and hKO-Iso). The number of TUNEL-positive myocytes was significantly higher in hKO-iso hearts 7 and 28 days after iso-infusion. The increase in myocyte cross-sectional area and fibrosis was higher in the WT-iso group. Matrix metalloproteinase-9 protein levels were significantly higher in WT-iso, whereas matrix metalloproteinase-2 levels were increased in hKO-iso hearts. Iso-infusion increased phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in both groups. The increase in c-Jun N-terminal kinase phosphorylation was significantly higher in hKO-iso (P<0.001 versus WT-iso). Thus, beta1 integrins play a crucial role in beta-adrenergic receptor-stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.

Myocardial extracellular matrix remodeling in ischemic heart failure.

Left ventricular (LV) remodeling is a process whereby structural alterations attempt to compensate altered hemodynamic load. In the chronic setting this process becomes maladaptive, self-sustaining and is associated with worsened survival. The extracellular matrix (ECM) of the heart, once believed an inert scaffold for cardiomyocytes, is now known to play an important role in LV remodeling. The enzyme system primarily responsible for ECM turnover is the matrix metalloproteinases (MMPs), and these enzymes are robustly altered in cardiovascular pathologies, including myocardial infarction (MI) and ischemic heart failure. A cause-and-effect relationship has been established between MMPs and LV remodeling post MI, as MMP inhibition prevents LV dilation and preserves cardiac function in animal models of infarction. In spite of this, initial clinical experience with MMP inhibition post MI has been disappointing. This review examines the structural and functional roles of the myocardial ECM, the evidence for MMP involvement in LV remodeling, and recent investigations into MMPs as prognostic markers and therapeutic targets.

Improvement of peripheral endothelial dysfunction by protein tyrosine phosphatase inhibitors in heart failure.

BACKGROUND: Chronic heart failure (CHF) induces endothelial dysfunction characterized by a decrease in nitric oxide (NO) production in response to flow (flow-mediated dilatation [FMD]). Because activation of endothelial NO synthase (eNOS) by flow requires tyrosine phosphorylation, we tested whether endothelial dysfunction could be corrected by increasing phosphotyrosine levels using protein tyrosine phosphatase (PTP) inhibitors and especially inhibitors of PTP1B. METHODS AND RESULTS: CHF was induced by coronary ligation in mice, and FMD was assessed in isolated and cannulated mesenteric artery segments (2 mm in length and <300 microm in diameter). CHF almost abolished FMD but only moderately affected the response to acetylcholine. In mice with CHF, the PTP1B inhibitors AS279, AS098, and AS713 restored FMD to levels similar to those of normal mice. This restoration was reduced by inhibitors of eNOS and phosphatidylinositol-3 kinase. Polymerase chain reaction and Western blot showed that arteries express PTP1B, and this expression was not affected by CHF. Immunolocalization revealed the presence of PTP1B in the endothelium and the adventitia. Flow induced a transient eNOS phosphorylation that was absent in CHF. PTP1B inhibition stimulated early eNOS phosphorylation and increased phosphorylation of Akt. CONCLUSIONS: Our results demonstrate for the first time that PTP1B inhibitors may be potent treatments for endothelial dysfunction.

Activation of the poly(ADP-ribose) polymerase pathway in human heart failure.

Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of acute and chronic myocardial dysfunction and heart failure. The goal of the present study was to investigate PARP activation in human heart failure, and to correlate PARP activation with various indices of apoptosis and oxidative and nitrosative stress in healthy (donor) and failing (NYHA class III-IV) human heart tissue samples. Higher levels of oxidized protein end-products were found in failing hearts compared with donor heart samples. On the other hand, no differences in tyrosine nitration (a marker of peroxynitrite generation) were detected. Activation of PARP was demonstrated in the failing hearts by an increased abundance of poly-ADP ribosylated proteins. Immunohistochemical analysis revealed that PARP activation was localized to the nucleus of the cardiomyocytes from the failing hearts. The expression of full-length PARP-1 was not significantly different in donor and failing hearts. The expression of caspase-9, in contrast, was significantly higher in the failing than in the donor hearts. Immunohistochemical analysis was used to detect the activation of mitochondrial apoptotic pathways. We found no significant translocation of apoptosis-inducing factor (AIF) into the nucleus. Overall, the current data provide evidence of oxidative stress and PARP activation in human heart failure. Interventional studies with antioxidants or PARP inhibitors are required to define the specific roles of these factors in the pathogenesis of human heart failure.

Activation of apoptotic caspase cascade during the transition to pressure overload-induced heart failure.

OBJECTIVES: A pressure overload model was developed to simulate aortic stenosis and assess caspase activity during the transition to heart failure. BACKGROUND: Cardiomyocyte apoptosis is implicated in the pathogenesis of heart failure, and caspase activation is central to this pathophysiological process. METHODS: A total of 10 sheep were banded with variable aortic constriction devices, progressively inflated to increase left ventricular (LV) afterload. Serial LV endomyocardial biopsy samples were obtained to measure caspase activity and presence of apoptosis. RESULTS: Over the first 3 to 4 weeks, hypertrophy developed in the sheep (LV mass index 90.8 +/- 4.9 g/m2 vs. 44.0 +/- 3.0 g/m2, p < 0.01), followed by gradual dilatation of the left ventricle (diastolic LV internal diameter 4.23 +/- 0.08 cm vs. 3.39 +/- 0.07 cm, p < 0.01). Ventricular function remained stable until 7 to 8 weeks after banding, when there was significant deterioration (fractional shortening 18.3 +/- 2.4% vs. 46.9 +/- 2.6%, p < 0.01), associated with clinical heart failure. Serial LV endomyocardial biopsy samples were obtained at each echocardiographically defined stage (LV hypertrophy, LV dilation, and LV failure). Activity of caspases-3, -8, and -9 (measured by specific fluorogenic peptide substrates and immunohistochemistry) increased progressively, particularly with the onset of myocardial dysfunction (caspase-3 7.92 +/- 1.19 vs. 1.00 +/- 0.15, caspase-8 1.94 +/- 0.21 vs. 1.00 +/- 0.04, caspase-9 5.87 +/- 0.97 vs. 1.00 +/- 0.18 relative fluorescent units, p < 0.05). No evidence of deoxyribonucleic acid (DNA) fragmentation, however, was identified by immunohistochemical assays. CONCLUSIONS: Activation of cardiomyocyte caspase enzymes occurs during the transition to heart failure, without completion of apoptotic DNA fragmentation. Increased activity of caspase-8 and -9 suggests both mitochondrial and death-receptor mediated pathways are involved in this pathological process. Further knowledge of these pathways may stimulate development of apoptosis-based strategies for slowing progression of heart failure in aortic stenosis patients.

Altered creatine kinase adenosine triphosphate kinetics in failing hypertrophied human myocardium.

BACKGROUND: The progression of pressure-overload left ventricular hypertrophy (LVH) to chronic heart failure (CHF) may involve a relative deficit in energy supply and/or delivery. METHODS AND RESULTS: We measured myocardial creatine kinase (CK) metabolite concentrations and adenosine triphosphate (ATP) synthesis through CK, the primary energy reserve of the heart, to test the hypothesis that ATP flux through CK is impaired in patients with LVH and CHF. Myocardial ATP levels were normal, but creatine phosphate levels were 35% lower in LVH patients (n = 10) than in normal subjects (n = 14, P < 0.006). Left ventricular mass and CK metabolite levels in LVH were not different from those in patients with LVH and heart failure (LVH+CHF, n = 10); however, the myocardial CK pseudo first-order rate constant was normal in LVH (0.36 +/- 0.04 s(-1) in LVH versus 0.32 +/- 0.06 s(-1) in normal subjects) but halved in LVH+CHF (0.17 +/- 0.06 s(-1), P < 0.001). The net ATP flux through CK was significantly reduced by 30% in LVH (2.2 +/- 0.7 micromol x g(-1) x s(-1), P = 0.011) and by a dramatic 65% in LVH+CHF (1.1 +/- 0.4 micromol x g(-1) x s(-1), P < 0.001) compared with normal subjects (3.1 +/- 0.8 micromol x g(-1) x s(-1)). CONCLUSIONS: These first observations in human LVH demonstrate that it is not the relative or absolute CK metabolite pool sizes but rather the kinetics of ATP turnover through CK that distinguish failing from nonfailing hypertrophic hearts. Moreover, the deficit in ATP kinetics is similar in systolic and nonsystolic heart failure and is not related to the severity of hypertrophy but to the presence of CHF. Because CK temporally buffers ATP, these observations support the hypothesis that a deficit in myofibrillar energy delivery contributes to CHF pathophysiology in human LVH.

Neuronal angiotensin II type 1 receptor upregulation in heart failure: activation of activator protein 1 and Jun N-terminal kinase.

Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT(1)R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT(1)R in the setting of CHF is not well understood. This study investigated the AT(1)R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT(1)R mRNA and protein expressions, plasma Ang II, and AP-1-DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4+/-30.2%, P<0.01; 206.6+/-25.8%, P<0.01; 280+/-36.5%, P<0.05; 207+/-16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9+/-48.1%, 213.8+/-27.7%, 148.2+/-10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II-induced AT(1)R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT(1)R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function.

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.

Arginase modulates myocardial contractility by a nitric oxide synthase 1-dependent mechanism.

Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate (l-arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes l-arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg II alone was found in isolated myocytes. Arg inhibition with S-(2-boronoethyl)-l-cysteine (BEC) augmented Ca(2+)-dependent NOS activity and NO production in myocytes, which did not depend on extracellular l-arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. Isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N-hydroxy-nor-l-arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N(G)-nitro-l-arginine methyl ester and S-methyl-l-thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg II negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.

Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression.

Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium "leak" via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activation of the "fight or flight" stress response leads to protein kinase A (PKA) hyperphosphorylation of RyR2 at Ser-2808. PKA phosphorylation of RyR2 Ser-2808 reduces the binding affinity of the channel-stabilizing subunit calstabin2, resulting in leaky RyR2 channels. We developed RyR2-S2808A mice to determine whether Ser-2808 is the functional PKA phosphorylation site on RyR2. Furthermore, mice in which the RyR2 channel cannot be PKA phosphorylated were relatively protected against the development of heart failure after myocardial infarction. Taken together, these data show that PKA phosphorylation of Ser-2808 on the RyR2 channel appears to be a critical mediator of progressive cardiac dysfunction after myocardial infarction.

Metalloproteinase inhibitor counters high-energy phosphate depletion and AMP deaminase activity enhancing ventricular diastolic compliance in subacute heart failure.

Cardiac matrix metalloproteinases (MMPs) stimulated by the sympathomimetic action of angiotensin II (AII) exacerbate chamber diastolic stiffening in models of subacute heart failure. Here we tested the hypothesis that MMP inhibition prevents such stiffening by favorably modulating high-energy phosphate (HEP) stores more than by effects on matrix remodeling. Dogs were administered AII i.v. for 1 week with tachypacing superimposed in the last two days (AII+P; n = 8). A second group (n = 9) underwent the same AII+P protocol but was preceded by oral treatment with an MMP inhibitor PD166793 [(S)-2-(4-bromo-biphenyl-4-sulfonylamino-3-methyl butyric acid] 1 week before and during the AII+P period. Pressure-volume analysis was performed in conscious animals, and myocardial tissue was subjected to in vitro and in situ zymography, collagen content, and HEP analysis (high-performance liquid chromatography). As reported previously, AII+P activated MMP9 and MMP2 and specifically exacerbated diastolic stiffening (+130% in chamber stiffness). PD166793 cotreatment prevented these changes, although myocardial collagen content, subtype, and cross-linking were unaltered. AII+P also reduced ATP, free energy of ATP hydrolysis (DeltaG(ATP)), and phosphocreatine while increasing free [ADP], AMP catabolites (nucleoside-total purines), and lactate. PD166793 reversed most of these changes, in part due to its inhibition of AMP deaminase. MMP activation may influence cardiac diastolic function by mechanisms beyond modulation of extracellular matrix. Interaction between MMP activation and HEP metabolism may play an important role in mediating diastolic dysfunction. Furthermore, these data highlight a potential major role for increased AMP deaminase activity in diastolic dysfunction.

Xanthine oxidase inhibition for chronic heart failure: is allopurinol the next therapeutic advance in heart failure?

A substantial body of evidence has accumulated to suggest a role for the xanthine oxidase metabolic pathway in the pathophysiology of chronic heart failure and other cardiovascular diseases.

Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats.

UNLABELLED: In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca2+-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatt's one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol 32P/min/mg non-collagen protein) was assessed in LV homogenates using 32P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. RESULTS: rats with chronic renal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2+/-2 versus 57.5+/-1; p < 0.05) and INH-2 activity (3.8+/-0.4 versus 36.2+/-4; p < 0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32+/-0.1 versus 0.73+/-0.04; p < 0.05) whereas that of phosphorylated INH-2 increased (3.05+/-0.3 versus 1.42+/-0.1; p < 0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59+/-0.05 versus 0.61+/-0.01; p < 0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77+/-0.05 versus 0.20+/-0.02; p < 0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts.

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.