Georges E. Haddad

β2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. We demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-β2AR complex could represent a hitherto unknown target for therapeutic intervention.

The Good, the Bad, and the Ugly with Alcohol Use and Abuse on the Heart

Since its advent, alcohol has been utilized throughout history socially, for rituals, worship, and for its therapeutic, antibacterial, and analgesic properties. In moderation, alcohol consumption and its use are generally viewed as clinically beneficial. Excessive alcohol consumption on the other hand has been recognized as having several adverse implications. Excessive use increases the risk of liver and heart disease, metabolic disturbances, nutritional deficiencies, certain cancers, brain damage, dementia, neuropathy, as well as other facets of morbidity and mortality. This review targets the sequelae of alcohol consumption on the heart, specifically on myocardial contractility, calcium channel signaling, and intracellular signaling pathways. With the incidence of alcohol-induced cardiac abnormalities being higher than previously thought, it is of increasing importance to elucidate the mechanisms behind them. Here, the cardiac effects of alcohol were not discussed in isolation but in conjunction with other important factors, such as high- and low-density lipoprotein levels and vascular dilatory influences. We explore these mechanisms, in particular, the oxidative stress as the major contributor, as well as pathways that may prove to be cardioprotective. As such, we demonstrate the involvement of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) as well as AKT that act as regulators of oxidative balance during oxidative stress responses. Thus, alcohol consumption may confer a cardioprotective effect when used in moderation through an AKT/NRF2-dependent mechanism.

Human cardiac-specific cDNA array for idiopathic dilated cardiomyopathy: sex-related differences

Idiopathic dilated cardiomyopathy (IDCM) constitutes a large portion of patients with heart failure of unknown etiology. Up to 50% of all transplant recipients carry this clinical diagnosis. Female-specific gene expression in IDCM has not been explored. We report sex-related differences in the gene expression profile of ventricular myocardium from patients undergoing cardiac transplantation. We produced and sequenced subtractive cDNA libraries, using human left ventricular myocardium obtained from male transplant recipients with IDCM and nonfailing human heart donors. With the resulting sequence data, we generated a custom human heart failure microarray for IDCM containing 1,145 cardiac-specific oligonucleotide probes. This array was used to characterize RNA samples from female IDCM transplant recipients. We identified a female gene expression pattern that consists of 37 upregulated genes and 18 downregulated genes associated with IDCM. Upon functional analysis of the gene expression pattern, deregulated genes unique to female IDCM were those that are involved in energy metabolism and regulation of transcription and translation. For male patients we found deregulation of genes related to muscular contraction. These data suggest that 1) the gene expression pattern we have detected for IDCM may be specific for this disease and 2) there is a sex-specific profile to IDCM. Our observations further suggest for the first time ever novel targets for treatment of IDCM in women and men.

Cardiorespiratory function associated with dietary nitrate supplementation

The advent of medical nutrition therapy and nutritional physiology affords the opportunity to link diet to specific cardiovascular mechanisms, suggesting novel treatments for cardiovascular disease. This study tests the hypothesis that beetroot juice increases the plasma nitric oxide (NO) concentration, which is associated with improvements in cardiorespiratory function at rest and during submaximal aerobic exercise. The subjects were 12 healthy, young adult, normotensive African-American females, with a body mass of 61 ± 2 kg, body fat of 28% ± 4%, and peak oxygen consumption of 26 ± 3 mL·kg(-1)·min(-1). The subjects were studied at rest and during cycle ergometer exercise at 40%, 60%, and 80% of peak oxygen consumption. Plasma NO concentration, respiratory quotient (RQ), minute ventilation, systolic and diastolic blood pressure (SBP and DBP), heart rate, and oxygen consumption were compared between isocaloric, isovolumetric placebo control orange juice and experimental beetroot juice treatments on separate days. The beetroot juice treatment increased plasma NO concentration and decreased oxygen consumption, SBP, and the heart rate-SBP product at rest and at 40%, 60%, and 80% of peak oxygen consumption in the absence of significant effects on RQ, minute ventilation, heart rate, and DBP. These findings suggest that, in healthy subjects, beetroot juice treatments increase plasma NO concentration and decrease cardiac afterload and myocardial oxygen demand at rest and during 3 submaximal levels of aerobic exercise. Future studies should determine the cellular and molecular mechanisms responsible for the improvement in cardiorespiratory function associated with dietary nitrate supplementation and whether they translate into better cardiovascular function and exercise tolerance in individuals with a compromised cardiovascular system.

Regulation of L-type inward calcium channel activity by captopril and angiotensin II via the phosphatidyl inositol 3- kinase pathway in cardiomyocytes from volume-overload hypertrophied rat hearts

Heart failure can be caused by pro-hypertrophic humoral factors such as angiotensin II (Ang II), which regulates protein kinase activities. The intermingled responses of these kinases lead to the early compensated cardiac hypertrophy, but later to the uncompensated phase of heart failure. We have shown that although beneficial, cardiac hypertrophy is associated with modifications in ion channels that are mainly mediated through mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) activation. This study evaluates the control of L-type Ca(2+) current (I(Ca,L)) by the Ang II/PI3K pathway in hypertrophied ventricular myocytes from volume-overload rats using the perforated patch-clamp technique. To assess activation of the I(Ca,L) in cardiomyocytes, voltages of 350 ms in 10 mV increments from a holding potential of -85 mV were applied to cardiocytes, with a pre-pulse to -45 mV for 300 ms. Volume overload-induced hypertrophy reduces I(Ca,L), whereas addition of Ang II alleviates the hypertrophic-induced decrease in a PI3K-dependent manner. Acute administration of Ang II (10(-6) mol/L) to normal adult cardiomyocytes had no effect; however, captopril reduced their basal I(Ca,L). In parallel, captopril regressed the hypertrophy and inverted the Ang II effect on I(Ca,L) seemingly through a PI3K upstream effector. Thus, it seems that regression of cardiac hypertrophy by captopril improved I(Ca,L) partly through PI3K.

Basal and IGF-I-dependent regulation of potassium channels by MAP kinases and PI3-kinase during eccentric cardiac hypertrophy

The potassium channels I(K) and I(K1), responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on I(K) and I(K1) through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10(-8) M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. I(K) and I(K1) activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal I(K) activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of I(K1) was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on I(K) and I(K1). We conclude that basal I(K) and I(K1) are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt.

Acute alcohol modulates cardiac function as PI3K/Akt regulates oxidative stress.

BACKGROUND Clinical manifestations of alcohol abuse on the cardiac muscle include defective contractility with the development of heart failure. Interestingly, low alcohol consumption has been associated with reduced risk of cardiovascular disease. Although several hypotheses have been postulated for alcoholic cardiomyopathy and for the low-dose beneficial cardiovascular effects, the precise mechanisms and mediators remain largely undefined. We hypothesize that modulation of oxidative stress by PI3K/Akt plays a key role in the cardiac functional outcome to acute alcohol exposure. METHODS Thus, acutely exposed rat cardiac tissue and cardiocytes to low (LA: 5 mM), moderate (MA: 25 mM), and high (HA: 100 mM) alcohol were assessed for markers of oxidative stress in the presence and absence of PI3K/Akt activators (IGF-1 0.1 μM or constitutively active PI3K: Ad.BD110 transfection) or inhibitor (LY294002 1 μM or Akt-negative construct Ad.Akt(K179M) transfection). RESULTS Acute LA reduced Akt, superoxide dismutase (SOD-3) and NFκB, ERK1, and p38 MAPK gene expression. Acute HA only increased that of SOD-3 and NFκB. These effects were generally inhibited by Ad.Akt(K179M) and enhanced with Ad.BD110 transfection. In parallel, LA reduced but HA enhanced Akt activity, which was reversed by IGF-1 and inhibited by Ad.Akt(K179M), respectively. Also, LA reduced caspase 3/7 activity and oxidative stress, while HA increased both. The former was blocked, while the latter effect was enhanced by Ad.Akt(K179M). The reverse was true with PI3K/Akt activation. This translated into reduced viability with HA, with no effect with LA. On the functional level, acute LA improved cardiac output and ejection fraction, mainly through increased stroke volume. This was accompanied with enhanced end-systolic pressure-volume relationship and preload recruitable stroke work. Opposite effect was recorded for HA. LA and HA in vivo functional effects were alleviated by LY and enhanced by IGF-1 treatment. CONCLUSIONS Acute LA and HA seem to oppositely affect cardiac function through modulation of oxidative stress where PI3K/Akt plays a pivotal role.

Effects of Dietary Nitrates on Systemic and Cerebrovascular Hemodynamics

Cerebral blood flow dysregulation is often associated with hypertension. We hypothesized that a beetroot juice (BRJ) treatment could decrease blood pressure and cerebrovascular resistance (CVR). We subjected 12 healthy females to control and BRJ treatments. Cerebrovascular resistance index (CVRI), systolic blood pressure (SBP), total vascular resistance (TVR), and the heart rate-systolic pressure product (RPP) measured at rest and at two exercise workloads were lower after the BRJ treatment. CVRI, SBP, and RPP were lower without a lower TVR at the highest exercise level. These findings suggest improved systemic and cerebral hemodynamics that could translate into a dietary treatment for hypertension.

Inotropic and lusitropic effects of calcitonin gene-related peptide in the heart

Previous studies have demonstrated positive-inotropic effects of calcitonin gene-related peptide (CGRP), but the mechanisms remain unclear. Therefore, two experiments were performed to determine the physiological correlates of the positive-inotropic effects of CGRP. Treatments designed to antagonize the effects of physiologically active CGRP₁₋₃₇ included posttreatment with CGRP₈₋₃₇ and pretreatment with LY-294002 (LY, an inhibitor of phosphatidylinositol 3-kinase), 17β-estradiol (E), and progesterone (P) were also used to modulate the effects of CGRP₁₋₃₇. Experiment 1 was in vitro studies on sarcomeres and cells of isolated adult rat cardiac myocytes. CGRP₁₋₃₇, alone and in combination with E and P, decreased sarcomere shortening velocities and increased shortening percentages, effects that were antagonized by CGRP₈₋₃₇, but not by LY. CGRP₁₋₃₇ increased resting intracellular calcium ion concentrations and Ca(2+) influxes, effects that were also antagonized by both CGRP₈₋₃₇ and LY. Experiment 2 was in vivo studies on left ventricular pressure-volume (PV) loops. CGRP₁₋₃₇ increased end-systolic pressure, ejection fraction, and velocities of contraction and relaxation while decreasing stroke volume, cardiac output, stroke work, PV area, and compliance. After partial occlusion of the vena cava, CGRP₁₋₃₇ increased the slope of the end-systolic PV relationship. CGRP₈₋₃₇ and LY attenuated most of the CGRP-induced changes. These findings suggest that CGRP-induced positive-inotropic effects may be increased by treatments with estradiol and progesterone and inhibited by LY. The physiological correlates of CGRP-induced positive inotropy observed in rat sarcomeres, cells, and intact hearts are likely to reveal novel mechanisms of heart failure in humans.

Alcohol and Apoptosis: Friends or Foes?

Alcohol abuse causes 79,000 deaths stemming from severe organ damage in the United States every year. Clinical manifestations of long-term alcohol abuse on the cardiac muscle include defective contractility with the development of dilated cardiomyopathy and low-output heart failure; which has poor prognosis with less than 25% survival for more than three years. In contrast, low alcohol consumption has been associated with reduced risk of cardiovascular disease, however the mechanism of this phenomenon remains elusive. The aim of this study was to determine the significance of apoptosis as a mediating factor in cardiac function following chronic high alcohol versus low alcohol exposure. Adult rats were provided 5 mM (low alcohol), 100 mM (high alcohol) or pair-fed non-alcohol controls for 4–5 months. The hearts were dissected, sectioned and stained with cresyl violet or immunohistochemically for caspase-3, a putative marker for apoptosis. Cardiomyocytes were isolated to determine the effects of alcohol exposure on cell contraction and relaxation. High alcohol animals displayed a marked thinning of the left ventricular wall combined with elevated caspase-3 activity and decreased contractility. In contrast, low alcohol was associated with increased contractility and decreased apoptosis suggesting an overall protective mechanism induced by low levels of alcohol exposure.