Function In Heart Failure Research Articles

An Emerging Strategy of Gene Therapy for Cardiac Disease

Heart disease is the major cause of morbidity and mortality worldwide.1,2 The current therapeutic approaches for heart failure are limited because postnatal cardiomyocytes have little regenerative capacity. Therefore, a new strategy needs to be established to improve the cardiac function. Article, see p 1147 Gene therapy is one of the most attractive new therapeutic strategies. Several kinds of cardiac gene therapy have so far been reported. Jeffrey M. Isner’s group reported that the administration of plasmid vectors encoding cDNA of the 165-amino acid isoform of vascular endothelial growth factor induced angiogenesis in patients with ischemic heart disease and with ischemic limbs.3,4 The effect of vascular endothelial growth factor gene therapy is mediated by the paracrine of cytokines (Figure, A). Figure. Schematic presentation of 3 types of gene therapies. Another type of gene therapy targets cardiomyocytes. The expression and activity of sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) have been observed to decrease in cardiomyocytes in a failing heart, and the overexpression of SERCA2a could restore the cardiac function in heart failure by improving calcium handling in the cardiomyocytes (Figure, B). Clinical trials for SERCA2A gene therapy were conducted for patients with heart failure, and positive results have been reported.5,6 A new strategy for gene therapy …

Impact of Short-duration Adaptive Servo-ventilation Therapy on Cardiac Function in Patients with Heart Failure

We investigated whether short-duration adaptive servo-ventilation (ASV) therapy improves cardiac function in heart failure (HF) patients. Eighty-six consecutive HF patients were divided into three groups: group A patients underwent daily ASV therapy for a mean duration of>4 h; group B patients used ASV therapy for>1 to <4 h per day; and group C patients declined or could not have daily ASV therapy for>1 h. The frequency in ASV use did not significantly differ between groups A (79.3±19.2%) and B (70.9±17.4%).

EFFECT OF N-3 POLYUNSATURATED FATTY ACIDS ON LEFT VENTRICULAR FUNCTION IN PATIENTS WITH CHRONIC HEART FAILURE: A META-ANALYSIS OF RANDOMISED PLACEBO-CONTROLLED TRIALS

Objectivesthe objective of this study was to assess the effect of supplementation of n-3 PUFA on left ventricular ejective fraction (LVEF), as a measure of left ventricular systolic function in...

Abstract 472: Effect of Acetylcholinesterase Inhibition with Pyridostigmine on Hemodynamics, Autonomic and Cardiac Function in Heart Failure C57Bl/6J Mice.

We investigated the effects of pyridostigmine (PYR) on hemodynamics, autonomic and cardiac function in heart failure (HF) mice. Telemetry transmitters (DSI) were implanted into the carotid artery under isofluorane anesthesia. After recovery, basal arterial pressure (AP, mmHg) and heart rate (HR, bpm) were recorded and parasympathetic and sympathetic tone (Δbpm) was evaluated by means of methyl atropine and propranolol. After the basal recording, the mice were submitted to left coronary ligation (LCL) and mini pumps implantation (PYR: 3mg/kg/day). Control mice were implanted with empty mini pumps. One and four weeks after LCL, the recordings were repeated. LCL promoted a myocardial infarction of 10-20%. HF mice presented decreased systolic AP (SAP) (113±2 vs 120±2 basal) and increased HR (548±12 vs 489±20 basal) only in the 1st week (n=8). In addition, LCL decreased vagal tone in the 1st week (121±14 vs 177±9 basal), increased sympathetic tone in the 1st and 4th week (-41±6 and -49±10 vs -23±2 basal). PYR prevented the increase of HR (505±10 vs 495±7 basal) but did not affect the decrease of the SAP (113±3 vs 123±1 basal) in the 1st week (n=3). PYR prevented the increase in the sympathetic tone (-23±15 and -23±6 vs -17±10 basal), but did not affect the parasympathetic tone (218±30 vs 199±37 basal) throughout the study. Cardiac function was assessed in separated mice (n=23) under isofluorane anesthesia. HF mice exhibited a decreased ejection fraction (28±4 vs 45±1% sham LCL) and increased diastolic and systolic volume (31±2 and 24±3 vs 18±2 and 12±1μl sham LCL). PYR increased the ejection fraction (44±4%) and attenuated the increase of both volumes (24±4 and 14±3μl). In conclusion, PYR prevented the tachycardia, decreased sympathetic tone and protected the cardiac function during the development (4 weeks) of HF in mice.

Coordinated Regulation of Murine Cardiomyocyte Contractility by Nanomolar (−)-Epigallocatechin-3-Gallate, the Major Green Tea Catechin

Green tea polyphenolic catechins exhibit biological activity in a wide variety of cell types. Although reports in the lay and scientific literature suggest therapeutic potential for improving cardiovascular health, the underlying molecular mechanisms of action remain unclear. Previous studies have implicated a wide range of molecular targets in cardiac muscle for the major green tea catechin, (-)-epigallocatechin-3-gallate (EGCG), but effects were observed only at micromolar concentrations of unclear clinical relevance. Here, we report that nanomolar concentrations of EGCG significantly enhance contractility of intact murine myocytes by increasing electrically evoked Ca(2+) transients, sarcoplasmic reticulum (SR) Ca(2+) content, and ryanodine receptor type 2 (RyR2) channel open probability. Voltage-clamp experiments demonstrate that 10 nM EGCG significantly inhibits the Na(+)-Ca(2+) exchanger. Of importance, other Na(+) and Ca(2+) handling proteins such as Ca(2+)-ATPase, Na(+)-H(+) exchanger, and Na(+)-K(+)-ATPase were not affected by EGCG ≤ 1 μM. Thus, nanomolar EGCG increases contractility in intact myocytes by coordinately modulating SR Ca(2+) loading, RyR2-mediated Ca(2+) release, and Na(+)-Ca(2+) exchange. Inhibition of Na(+)-K(+)-ATPase activity probably contributes to the positive inotropic effects observed at EGCG concentrations >1 μM. These newly recognized actions of nanomolar and micromolar EGCG should be considered when the therapeutic and toxicological potential of green tea supplementation is evaluated and may provide a novel therapeutic strategy for improving contractile function in heart failure.

Abstract 123: SUMO1 Modification Regulates SR Calcium ATPase Pump, SERCA2a in Heart Failure

Background: The cardiac calcium ATPase, SERCA2a, is a critical pump responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. However, the underlying mechanisms of SERCA2a’s dysfunction remain incompletely understood. Methods and Results: In this study, we show that SERCA2a is modified by SUMO1 at lysine sites 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. SUMO1 and SERCA2a SUMOylation levels were both decreased in mouse and pig models of heart failure and failing human left ventricles. To determine whether reduced SUMO1 levels are responsible for reduced SERCA2a protein levels and reduced cardiac function, we used an adenovirus associated virus-mediated gene delivery approach to up-regulate SUMO1 in trans aortic constriction-induced mouse model of heart failure. We found that increasing SUMO1 levels led to a restoration of SERCA2a levels, improved hemodynamic performance, and reduced mouse mortality. By contrast, down-regulation of SUMO1 using small hairpin RNA accelerated cardiac functional deterioration and was accompanied by decreased SERCA2a function. Conclusion: In this study, we study a new mechanism for modulation of SERCA2a activity and beneficial effects of SUMO1 in the setting of heart failure. It suggests that changes in post-translational modifications of SERCA2a could negatively affect cardiac function in heart failure. Our data may provide a new platform for the design of therapeutic strategies for heart failure.

A contemporary view on endothelial function in heart failure

The assessment of different aspects of endothelial dysfunction in cardiovascular medicine in general and in heart failure (HF) has been the focus of intense research, and includes vasomotor, haemostatic, antioxidant, and inflammatory activities. Differences also exist in the pattern of endothelial dysfunction depending on aetiology, severity, and stability of HF in individual patients. In the majority of patients with ischaemic aetiology of HF, endothelial dysfunction is systemic in its nature and involves both arteries and veins, conductance vessels and microvascular beds, coronary, pulmonary, and peripheral vessels. The pattern of endothelial dysfunction is more heterogeneous in non-ischaemic HF, with fewer features of systemic abnormalities. Indeed, many subjects with non-ischaemic HF have a functionally preserved endothelium in peripheral arteries, with endothelial dysfunction seen only in coronary vessels. Endothelial dysfunction has significant prognostic value in HF, but its clinical application is hampered by methodological limitations in its assessment. Various medications (including angiotensin-converting enzyme inhibitors and statins) and regular physical activity have been shown to improve endothelial function in HF. However, there are still no pharmaceutical agents specifically targeting the vascular endothelium. Despite the large number of studies, the pathophysiological role of the vascular endothelium and its clinical potential as a therapeutic target has not yet been sufficiently developed and undoubtedly awaits further exploration.

Role of platelets in NOX2 activation mediated by TNFα in heart failure

Tumor necrosis factor (TNF) α may contribute to the deterioration of cardiovascular function in heart failure (HF) through various mechanisms, including the generation of reactive oxygen species (ROS). NADPH oxidase is the major source of ROS in the vascular system, but the interplay between TNFα and NADPH oxidase activation is elusive. As platelets possess NADPH oxidase enzyme, they represent an important tool to investigate the interplay between NADPH oxidase and TNFα in patients with HF. Serum gp91phox (NOX2), the catalytic core of NADPH oxidase, and serum TNFα were measured in 120 HF patients and in 60 healthy subjects. Compared with healthy subjects, HF patients had higher blood levels of NOX2 and TNFα with a progressive increase from NYHA I to NYHA IV classes. NOX2 levels in blood were independently associated with TNFα in HF patients. An in vitro study, performed on platelets from a subgroup of HF patients, shows that TNFα, at concentrations commonly found in HF patients' peripheral circulation, activates platelet NOX2. Thus, TNFα increases ROS production and the extracellular levels of NOX2. These phenomena are inhibited by the NOX2-specific blocking peptide gp91ds-tat. The study provides evidence that circulating NOX2, as well as the activation of NOX2 on platelets, is increased in HF likely as a consequence of the underlying inflammatory process.

Serum uric acid as an index of impaired renal function in congestive heart failure

BackgroundHyperuricemia is frequently present in patients with heart failure. Many pathological conditions, such as tissue ischemia, renal function impairment, cardiac function impairment, metabolic syndrome, and inflammatory status, may impact uric acid (UA) metabolism. This study was to assess their potential relations to UA metabolism in heart failure.MethodsWe retrospectively assessed clinical characteristics, echocardiological, renal, metabolic and inflammatory variables selected on the basis of previous evidence of their involvement in cardiovascular diseases and UA metabolism in a large cohort of randomly selected adults with congestive heart failure (n = 553). By clustering of indices, those variables were explored using factor analysis.ResultsIn factor analysis, serum uric acid (SUA) formed part of a principal cluster of renal functional variables which included serum creatinine (SCr) and blood urea nitrogen (BUN). Univariate correlation coefficients between variables of patients with congestive heart failure showed that the strongest correlations for SUA were with BUN (r = 0.48, P < 0.001) and SCr (r = 0.47, P < 0.001).ConclusionsThere was an inverse relationship between SUA levels and measures of renal function in patients with congestive heart failure. The strong correlation between SUA and SCr and BUN levels suggests that elevated SUA concentrations reflect an impairment of renal function in heart failure.

Sleep Apnea and Cognitive Function in Heart Failure

Background. Prior research indicates that heart failure (HF) patients exhibit significant cognitive deficits on neuropsychological testing. Sleep apnea is associated with both HF and reduced cognitive function, but the combined impact of these conditions on cognitive function is unknown. Methods. In the current study, 172 older adults with a dual diagnosis of HF and sleep apnea or HF alone completed a battery of cognitive tests measuring attention, executive functioning, and memory. Results. Relative to patients with HF alone, persons with both HF and sleep apnea performed worse on measures of attention after adjusting for demographic and medical variables. Conclusions. The current findings suggest that HF patients with comorbid sleep apnea may be at greater risk for cognitive impairment relative to HF patient without such history. Further work is needed to clarify mechanisms for these findings and to determine whether the interactive effects on cognitive function lead to poorer patient outcomes.

Proximal Cerebral Arteries Develop Myogenic Responsiveness in Heart Failure via Tumor Necrosis Factor-α–Dependent Activation of Sphingosine-1-Phosphate Signaling

Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-α (TNFα)-dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNFα expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNFα antagonist etanercept. TNFα mediates its effect via a sphingosine-1-phosphate (S1P)-dependent mechanism, requiring sphingosine kinase 1 and the S1P(2) receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNFα/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Clinical Methods to Assess Cardiac Sympathetic Innervation and Function

Physiological, pharmacologic, neurochemical, and neuroimaging methods enable evaluation of cardiac sympathetic noradrenergic innervation and function in humans. This lecture provides examples of each approach. Low frequency power of heart rate variability, adjusted for total or high frequency power, has been used to examine cardiac sympathetic “tone” or “sympathovagal balance” but seems related more closely to baroreflex modulation of autonomic outflows than to the outflows themselves. Effects drugs on cardiac contractility can separate pre‐ from post‐ganglionic noradrenergic lesions. Measurement of norepinephrine and its main neuronal metabolite during infusion of tracer‐labeled norepinephrine provides a means to examine key aspects of cardiac sympathetic function in heart failure. Cardiac sympathetic imaging is very powerful for detecting dysfunction or loss of cardiac sympathetic nerves. Using this technology we described cardiac sympathetic denervation in Parkinson disease and obtained evidence for decreased vesicular uptake of intra‐neuronal catecholamines in Lewy body diseases. Clinical laboratory evaluation therefore can provide important clues to the pathophysiology of neurocardiologic disorders.

One Dose of Ghrelin Prevents the Acute and Sustained Increase in Cardiac Sympathetic Tone after Myocardial Infarction

Acute myocardial infarction (MI) increases sympathetic nerve activity (SNA) to the heart, which exacerbates chronic cardiac deterioration. The hormone ghrelin, if administered soon after an MI, prevents the increase in cardiac SNA and improves early survival prognosis. Whether these early beneficial effects of ghrelin also impact on cardiac function in chronic heart failure has not yet been addressed and thus was the aim of this study. MI was induced in Sprague Dawley rats by ligating the left coronary artery. One bolus of saline (n = 7) or ghrelin (150 μg/kg, sc, n = 9) was administered within 30 min of MI. Two weeks after the infarct (or sham; n = 7), rats were anesthetized and cardiac function was evaluated using a Millar pressure-volume conductance catheter. Cardiac SNA was measured using whole-nerve electrophysiological techniques. Untreated-MI rats had a high mortality rate (50%), evidence of severe cardiac dysfunction (ejection fraction 28%; P < 0.001), and SNA was significantly elevated (102% increase; P = 0.03). In comparison, rats that received a single dose of ghrelin after the MI tended to have a lower mortality rate (25%; P = NS) and no increase in SNA, and cardiac dysfunction was attenuated (ejection fraction of 43%; P = 0.014). This study implicates ghrelin as a potential clinical treatment for acute MI but also highlights the importance of therapeutic intervention in the early stages after acute MI. Moreover, these results uncover an intricate causal relationship between early and chronic changes in the neural control of cardiac function in heart failure.

Resistance training alters skeletal muscle structure and function in human heart failure: effects at the tissue, cellular and molecular levels

Reduced skeletal muscle function in heart failure (HF) patients may be partially explained by altered myofilament protein content and function. Resistance training increases muscle function, although whether these improvements are achieved by correction of myofilament deficits is not known. To address this question, we examined 10 HF patients and 14 controls prior to and following an 18 week high-intensity resistance training programme. Evaluations of whole muscle size and strength, single muscle fibre size, ultrastructure and tension and myosin-actin cross-bridge mechanics and kinetics were performed. Training improved whole muscle isometric torque in both groups, although there were no alterations in whole muscle size or single fibre cross-sectional area or isometric tension.Unexpectedly, training reduced the myofibril fractional area of muscle fibres in both groups. This structural change manifested functionally as a reduction in the number of strongly bound myosin-actin cross-bridges during Ca²⁺ activation. When post-training single fibre tension data were corrected for the loss of myofibril fractional area, we observed an increase in tension with resistance training. Additionally, training corrected alterations in cross-bridge kinetics (e.g. myosin attachment time) in HF patients back to levels observed in untrained controls. Collectively, our results indicate that improvements in myofilament function in sedentary elderly with and without HF may contribute to increased whole muscle function with resistance training. More broadly, these data highlight novel cellular and molecular adaptations in muscle structure and function that contribute to the resistance-trained phenotype.

Age-related effects of exercise training on diastolic function in heart failure with reduced ejection fraction: The Leipzig Exercise Intervention in Chronic Heart Failure and Aging (LEICA) Diastolic Dysfunction Study

Diastolic dysfunction (DD) was identified as a predictor of adverse prognosis in heart failure with reduced ejection fraction (HFREF). It is, however, unknown if DD is improved by exercise training, which is known to induce reverse remodelling, and if the training effect is attenuated in elderly HFREF patients. We therefore assessed DD in a cohort of referent controls (RCs) and HFREF patients and studied the response of DD to endurance exercise in two age groups (≤55 years and ≥65 years). Sixty RC (30 ≤ 55 years, mean age 50 ± 5 years; 30 ≥ 65 years, 72 ± 4 years) and 60 HFREF patients (30 ≤ 55 years, 46 ± 5 years; 30 ≥ 65 years, 72 ± 5 years, EF 28 ± 5%) were randomized to 4 weeks of supervised endurance training or to a control group. Exercise training was effective in reducing LV isovolumetric relaxation time by 29% in young and by 26% in old HFREF patients (P< 0.05 for both). As assessed by tissue Doppler, septal E' increased by 37% in young and by 39% among old HFREF patients (P< 0.005 for both) resulting in a significant decrease in the E/E' ratio from 13 ± 1 to 10 ± 1 in young and 14 ± 1 to 11 ± 1 in old HFREF patients (P< 0.05 for both). Serum levels of N-terminal pro brain natriuretic peptide were significantly reduced after endurance training in HFREF patients of all ages. In HFREF, diastolic function is significantly impaired in all age groups. Endurance training is highly effective in improving left ventricular diastolic function in HFREF patients regardless of age. This study is registered at ClinicalTrials.gov (number: NCT00176319).

Structural Remodeling and Mechanical Function in Heart Failure

The cardiac extracellular matrix (ECM) is the three-dimensional scaffold that defines the geometry and muscular architecture of the cardiac chambers and transmits forces produced during the cardiac cycle throughout the heart wall. The cardiac ECM is an active system that responds to the stresses to which it is exposed and in the normal heart is adapted to facilitate efficient mechanical function. There are marked differences in the short- and medium-term changes in ventricular geometry and cardiac ECM that occur as a result of volume overload, hypertension, and ischemic cardiomyopathy. Despite this, there is a widespread view that a common remodeling "phenotype" governs the final progression to end-stage heart failure in different forms of heart disease. In this review article, we make the case that this interpretation is not consistent with the clinical and experimental data on the topic. We argue that there is a need for new theoretical and experimental models that will enable stresses acting on the ECM and resultant deformations to be estimated more accurately and provide better spatial resolution of local signaling mechanisms that are activated as a result. These developments are necessary to link the effects of structural remodeling with altered cardiac mechanical function.

Effect of Short-Duration Adaptive Servo-Ventilation Therapy on Cardiac Function in Patients With Heart Failure

The aim of this study was to investigate whether short-duration adaptive servo-ventilation (ASV) therapy improves cardiac function in heart failure (HF) patients. Consecutive HF patients (n=86) were divided into 3 groups: group A, ASV for a mean of ≥4 h; group B, ASV for ≥1 to <4 h per day; and group C, no ASV or ASV <1 h. The frequency of ASV use did not significantly differ between groups A (79.3±19.2%) and B (70.9±17.4%). After 6 months, a significant increase in left ventricular ejection fraction (LVEF), significant decrease in plasma brain natriuretic peptide (BNP) and decrease in LV end-diastolic volume (LVEDV) were observed in groups A (LVEF, 5.0±8.1%; BNP, -24.9±33.7%; LVEDV, -6.2±10.1%) and B (LVEF, 3.5±5.5%; BNP, -16.5±24.6%; LVEDV, -5.1±8.2%) as compared with group C (LVEF, -1.5±6.0%, P=0.004, P=0.017; BNP, 2.8±10.2%, P=0.002, P=0.017; LVEDV, 0.8±9.1%, P=0.031, P=0.043). Significant correlation was seen between the total ASV time and changes of LVEF (r=0.369, P=0.002), BNP (r=-0.445, P<0.001), and LVEDV (r=-0.374, P=0.001). Admission rate was lower in groups A (4.1%) and B (7.1%) than in group C (25%, log-rank test; P=0.042, P=0.045). Multivariate analysis showed that the frequency of ASV use was a strong parameter for the improvement of LVEF (coefficient=0.284, standard error=0.035, P=0.019). Even a short-duration of ASV therapy may improve cardiac function in HF patients.

G i -Biased β 2 AR Signaling Links GRK2 Upregulation to Heart Failure

Phosphorylation of β(2)-adrenergic receptor (β(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of β(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to βAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant β(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type β(2)AR (WT-TG) or a mutant β(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased β(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of β(2)AR by GRK and resultant increase in G(i)-biased β(2)AR signaling play an important role in the development of heart failure. Our data show that enhanced β(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased β(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Abstract P131: An Akt-Phosphomimetic Sequence of the Cavb2 C-Terminal Region Protects L-Type Calcium Channels from Protein Degradation

Alteration in the density or function of L-Type Calcium Channels (LTCCs) has been related to cardiovascular diseases such as heart failure and diabetic cardiomyopathy. It could therefore be envisioned that increasing LTCC density may improve cardiac function in heart failure. Recently, we determined that the Ser-Thr kinase Akt plays a key role in regulating cardiac inotropism through the modulation of LTCC density and function. Specifically, we found that the LTCC pore-forming channel subunit Cava1.2 contains highly evolutionary conserved PEST sequences (signals for rapid proteolytic degradation) that are responsible for direct Cava1.2 protein degradation. Phosphorylation of the C-terminal coiled coil of the Cavb2 chaperone subunit enhances LTCC protein stability by preventing PEST-mediated Cava1.2 degradation. The aim of this study was to further dissect this Akt-dependent fine-tuning mechanism regulating LTCC density, searching for potential Akt-phosphomimetic (APM) molecules that could enhance LTCC density. Using yeast two-hybrid screening, we found that APM Cavb2 sequences interact with the globular domain of Cavb2 itself in a solvent-exposed region that we named Tail Interacting Domain (TID). Biochemical and functional assays as well as site-specific mutagenesis in TID identified the minimal aminoacid sequence responsible for the TID-tail interaction. In addition, through an approach comprising western blot analyses, fluorescent-based calcium assays, calcium current (ICaL) measurements, molecular modeling and peptide arrays, we identified the minimal APMs that efficiently protects Cava1.2 from protein degradation. Based on our in vitro results, we suggest that the identified APM sequence/peptide could be used as a “therapeutic approach”; for increasing or reestablishing impaired cardiac contractility in mouse models of cardiomyopathy in which the LTCC density is altered, thus enhancing inotropism.

Abstract 18196: Aldehyde Oxidase is a Potent Source of Superoxide Generation and Can Modulate Cardiac Contractile Function

Aldehyde oxidase (AO) is a cytosolic enzyme belonging to the xanthine oxidoreductase (XOR) family. A variety of aldehyde substrates are known to be formed in cardiovascular disease secondary to inflammation and oxidative stress that are efficient AO substrates. While XOR has been reported to influence heart function in heart failure and is well known to be an important source of superoxide (O 2 • - ), little is known regarding the role of AO in cardiac oxidant signaling. In order to assess the presence of AO in the heart and its potential role in cardiovascular pathophysiology, we studied the distribution of AO in rat myocardium and isolated cardiac myocytes as well as its levels and activities in heart tissue. Furthermore, in isolated perfused rat hearts we measured the functional effect of AO activation or inhibition. Immunohistochemistry, using an AO specific antibody, showed a myofibrillar / sarcomere distribution for AO. In control rat myocardium, the activity of AO was found to be 1.5-fold higher than that of total XOR (0.21±0.02 vs . 0.14±0.01 nmol min -1 mg -1 of protein), and the levels of protein 2.5-fold higher than that of XOR (30.26±2.42 vs . 11.86±1.07 nM), while O 2 • - production (measured by cytochrome c assay) in the presence of optimal substrate levels was estimated to be &gt;10-fold higher for AO than XOR. In isolated perfused rat hearts, infusion with the AO substrate 4-(dimethylamino)cinnamaldehyde ( p -DMAC) (10 μM) resulted in a significant increase in left ventricular developed pressure (LVDP) of 14±3% (p&lt;0.05), while treatment with the potent inhibitor amadone (10 μM) completely inhibited this increase. Thus, AO is present in cardiac myocytes adjacent to the contractile proteins and in the presence of aldehyde substrates it can function as an important source of O 2 • - modulating contractile function. Further studies will be needed to assess the potential role of AO in modulating cardiac function in ischemic syndromes and heart failure.