Crosstalk between leptin and interleukin-1ß abrogates negative inotropic effects in a model of chronic hyperleptinemia.

Interleukin 1 beta (IL-1ß) is a proinflammatory cytokine with potent cardiosuppressive effects. Previous studies have shown that leptin blunts the negative inotropic effects of IL-1ß in isolated adult rat cardiac myocytes. However, the interactions between leptin and IL-1ß in the heart have not been examined on a background of chronic hyperleptinemia. To study this interaction, we have chosen the SHHF rat, a model of spontaneous hypertension that ultimately develops congestive heart failure. SHHF that are heterozygous for a null mutation of the leptin receptor (+/fa(cp), HET) are phenotypically lean but chronically hyperleptinemic and develop heart failure earlier than their normoleptinemic true lean (+/+, LN) littermates. Simultaneous cell shortening and calcium transients were measured in isolated ventricular cardiac myocytes from LN and HET SHHF in response to leptin, IL-1ß or IL-1ß following one hour pretreatment with leptin. Despite evidence of metabolic leptin resistance, HET myocytes were sensitive to the negative inotropic effect of leptin, similar to LN. Contractility returned to control levels in myocytes from HET that were pretreated with leptin prior to IL-1ß, while contractility remained depressed compared with control and similar to leptin alone in LN. Chronic hyperleptinemia resulted in altered JAK/STAT signaling in response to leptin and IL-1ß in isolated perfused hearts from HET compared with LN SHHF. Phosphorylated STAT3 (pSTAT3) and STAT5 (pSTAT5) decreased when HET hearts were treated with leptin followed by IL-1ß. While decreases in pSTAT3 and pSTAT5 may be associated with abrogation of the acute negative inotropic effects of IL-1ß in the presence of leptin in HET, long-term consequences remain to be explored. This study demonstrates that the heart remains sensitive to leptin in a hyperleptinemic state. Crosstalk between leptin and IL-1ß can influence cardiac function and cytokine signaling and these interactions are moderated by the presence of long-term hyperleptinemia.

Leptin modulates the negative inotropic effect of interleukin-1beta in cardiac myocytes.

Interleukin-1beta (IL-1beta) is a potent negative inotrope implicated in the functional abnormalities of heart failure. Because the adipokine, leptin, protects against some of the cardiovascular effects of endotoxin, we hypothesized that leptin may modulate the cardiosuppressive effects of IL-1beta in isolated cardiomyocytes. Ventricular cardiac myocytes isolated from adult male Sprague Dawley rats were analyzed simultaneously for electrically stimulated contractility and calcium transients following 30 min exposure to IL-1beta (10 ng/ml) with or without 60 min pretreatment with leptin (25 ng/ml). IL-1beta decreased cell shortening, depressed maximal velocities of shortening and relengthening, and prolonged the time to 90% relaxation. The change in fura2-AM fluorescence ratio amplitude (Delta[Ca(2+)]) was significantly depressed and the time to return to baseline [Ca(2+)] was prolonged. The negative inotropic effects of IL-1beta were blocked by the neutral sphingomyelinase inhibitor Manumycin A (5 microM) or the ceramidase inhibitor N-oleoyl ethanolamine (1 microM). Prior exposure of myocytes to leptin blocked IL-1beta-induced cardiosuppression in conjunction with a blunting of IL-1beta stimulated ceramide accumulation. These data suggest that leptin may modulate IL-1beta signaling through the sphingolipid signaling pathway in cardiomyocytes.

Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation.

Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.

Cardiac dysfunction in the R6/2 mouse model of Huntington's disease.

Recent evidence suggests that mutant huntingtin protein-induced energetic perturbations contribute to neuronal dysfunction in Huntington's disease (HD). Given the ubiquitous expression of huntingtin, other cell types with high energetic burden may be at risk for HD-related dysfunction. Early-onset cardiovascular disease is the second leading cause of death in HD patients; a direct role for mutant huntingtin in this phenomenon remains unevaluated. Here we tested the hypothesis that expression of mutant huntingtin is sufficient to induce cardiac dysfunction, using a well-described transgenic model of HD (line R6/2). R6/2 mice developed cardiac dysfunction by 8 weeks of age, progressing to severe failure at 12 weeks, assessed by echocardiography. Limited evidence of cardiac remodeling (e.g. hypertrophy, fibrosis, apoptosis, beta(1) adrenergic receptor downregulation) was observed. Immunogold electron microscopy demonstrated significant elevations in nuclear and mitochondrial polyglutamine presence in the R6/2 myocyte. Significant alterations in mitochondrial ultrastructure were seen, consistent with metabolic stress. Increased cardiac lysine acetylation and protein nitration were observed and were each significantly associated with impairments in cardiac performance. These data demonstrate that mutant huntingtin expression has potent cardiotoxic effects; cardiac failure may be a significant complication of this important experimental model of HD. Investigation of the potential cardiotropic effects of mutant huntingtin in humans may be warranted.

Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats.

Hypertension is a common cause of heart failure, and ventricular arrhythmias are a major cause of death in heart failure. The spontaneous hypertension heart failure (SHHF) rat model was used to study altered ventricular electrophysiology in hypertension and heart failure. We hypothesized that a reduction in the inward rectifier K(+) current (I(K1)) and expression of pacemaker current (I(f)) would favor abnormal automaticity in the SHHF ventricle. SHHF ventricular myocytes were isolated at 2 and 8 mo of age and during end-stage heart failure (>/=17 mo); myocytes from age-matched rats served as controls. Inward I(K1) was significantly reduced at both 8 and >/=17 mo in SHHF rats compared with controls. There was a reduction in inward I(K1) due to aging in the controls only at >/=17 mo. We found a significant increase in I(f) at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in I(f). Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an I(f) blocker (10 muM zatebradine). Increased I(f) during hypertension alone or combined increases in I(f) with reduced I(K1) during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.

Endurance exercise training attenuates cardiac beta2-adrenoceptor responsiveness and prevents ventricular fibrillation in animals susceptible to sudden death.

Enhanced cardiac beta(2)-adrenoceptor (beta(2)-AR) responsiveness can increase susceptibility to ventricular fibrillation (VF). Exercise training can decrease cardiac sympathetic activity and could, thereby, reduce beta(2)-AR responsiveness and decrease the risk for VF. Therefore, dogs with healed myocardial infarctions were subjected to 2 min of coronary occlusion during the last minute of a submaximal exercise test; VF was observed in 20 susceptible, but not in 13 resistant, dogs. The dogs were then subjected to a 10-wk exercise-training program (n = 9 susceptible and 8 resistant) or an equivalent sedentary period (n = 11 susceptible and 5 resistant). Before training, the beta(2)-AR antagonist ICI-118551 (0.2 mg/kg) significantly reduced the peak contractile (by echocardiography) response to isoproterenol more in the susceptible than in the resistant dogs: -45.5 +/- 6.5 vs. -19.2 +/- 6.3%. After training, the susceptible and resistant dogs exhibited similar responses to the beta(2)-AR antagonist: -12.1 +/- 5.7 and -16.2 +/- 6.4%, respectively. In contrast, ICI-118551 provoked even greater reductions in the isoproterenol response in the sedentary susceptible dogs: -62.3 +/- 4.6%. The beta(2)-AR agonist zinterol (1 microM) elicited significantly smaller increases in isotonic shortening in ventricular myocytes from susceptible dogs after training (n = 8, +7.2 +/- 4.8%) than in those from sedentary dogs (n = 7, +42.8 +/- 5.8%), a response similar to that of the resistant dogs: +3.0 +/- 1.4% (n = 6) and +3.2 +/- 1.8% (n = 5) for trained and sedentary, respectively. After training, VF could no longer be induced in the susceptible dogs, whereas four sedentary susceptible dogs died during the 10-wk control period and VF could still be induced in the remaining seven animals. Thus exercise training can restore cardiac beta-AR balance (by reducing beta(2)-AR responsiveness) and could, thereby, prevent VF.

Calmodulin interactions with IQ peptides from voltage-dependent calcium channels.

Calmodulin (CaM) functions as a Ca(2+) sensor for inactivation and, in some cases, facilitation of a variety of voltage-dependent Ca(2+) channels. A crucial determinant for CaM binding to these channels is the IQ motif in the COOH-terminal tail of the channel-forming subunit. The binding of CaM to IQ peptides from Lc-, P/Q-, and R-type, but not N-type, voltage-dependent Ca(2+) channels increases the Ca(2+) affinity of both lobes of CaM, producing similar N- and C-lobe Ca(2+) affinities. Ca(2+) associates with and dissociates from the N-lobe much more rapidly than the C-lobe when CaM is bound to the IQ peptides. Compared with the other IQ peptides, CaM-bound Lc-IQ has the highest Ca(2+) affinity and the most rapid rates of Ca(2+) association at both lobes, which is likely to make Ca(2+) binding to CaM, bound to this channel, less sensitive than other channels to intracellular Ca(2+) buffers. These kinetic differences in Ca(2+) binding to the lobes of CaM when bound to the different IQ motifs may explain both the ability of CaM to perform multiple functions in these channels and the differences in CaM regulation of the different voltage-dependent Ca(2+) channels.

Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel.

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.

Sorcin regulates excitation-contraction coupling in the heart.

Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart.

Selective contractile dysfunction of left, not right, ventricular myocardium in the SHHF rat.

The progression of hypertension to cardiac failure involves systemic changes that may ultimately affect contractility throughout the heart. Spontaneous hypertensive heart failure (SHHF) rats have depressed left ventricular (LV) function, but right ventricular (RV) dysfunction is less well characterized. Ultrathin (87 +/- 5 mircom) trabeculae were isolated from end-stage failing SHHF rats and from age-matched controls. Under near-physiological conditions (1 mM Ca(2+), 37 degrees C, 4 Hz), developed force (in mN/mm(2)) was not significantly different in SHHF LV and RV trabeculae and those of controls. SHHF LV preparations displayed a negative force-frequency behavior (40 +/- 7 vs. 23 +/- 4 mN/mm(2), 2 vs. 7 Hz); this relationship was positive in SHHF RV preparations (27 +/- 5 vs. 40 +/- 6 mN/mm(2)) and controls (32 +/- 6 vs. 44 +/- 9 mN/mm(2)). The response to isoproterenol (10(-6) M, 4 Hz) was depressed in SHHF LV preparations. The inotropic response to hypothermia was lost in SHHF LV trabeculae but preserved in SHHF RV trabeculae. Intracellular calcium measurements revealed impaired calcium handling at higher frequencies in LV preparations. We conclude that in end-stage failing SHHF rats, RV function is only marginally affected, whereas a severe contractile dysfunction of LV myocardium is present.

Human T-cell lymphotropic virus type 1 p12(I) expression increases cytoplasmic calcium to enhance the activation of nuclear factor of activated T cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) establishes persistent infection and is associated with lymphoproliferative or neurodegenerative diseases. As a complex retrovirus, HTLV-1 contains typical structural and enzymatic genes, as well as regulatory and accessory genes encoded in the pX region. The early events necessary for HTLV-1 to establish infection in lymphocytes, its primary target cells, remain unresolved. Recent studies have demonstrated the importance of regulatory and accessory gene products in determining this virus-host interaction. Among these, pX open reading frame I, which encodes two proteins, p12(I) and p27(I), is required for establishing persistent infection in vivo and for infection in quiescent primary lymphocytes. In addition, p12(I) localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus and associates with a calcium binding protein, calreticulin. We recently reported that p12(I) expression induces the calcium-responsive T-cell transcription factor, nuclear factor of activated T cells (NFAT), in the presence of phorbol ester activation. Based on these studies, we hypothesize that p12(I) may modulate calcium release from the ER. Here, we report that p12(I) expression increases basal cytoplasmic calcium and concurrently diminishes calcium available for release from the ER stores. Overexpression of calreticulin, a calcium buffer protein, blocked p12(I)-mediated NFAT activation independently of its ability to bind p12(I). Chemical inhibition studies using inhibitors of inositol 1,4,5-triphosphate receptor and calcium release-activated calcium channels suggest that inositol 1,4,5-triphosphate receptor in the ER membrane and calcium release-activated calcium channels in the plasma membrane contribute to p12(I)-mediated NFAT activation. Collectively, our results are the first to demonstrate the role of p12(I) in elevating cytoplasmic calcium, an antecedent to T-cell activation, and further support the important role of this accessory protein in the early events of HTLV-1 infection.

Phase I and phase II of short-term mechanical restitution in perfused rat left ventricles.

We examined the contributions of the Ca(2+) channels of the sarcolemma and of the sarcoplasmic reticulum to electromechanical restitution. Extrasystoles (F(1)) were interpolated 40-600 ms following a steady-state beat (F(0)) in perfused rat ventricles paced at 2 or 3 Hz. Plots of F(1)/F(0) versus the extrasystolic interval consisted of phase I, which occurred before relaxation of the steady-state beat, and phase II, which occurred later. Phase I exhibited a period of enhanced left ventricular pressure development that coincided with action potential prolongation. Phase I was eliminated by -BAY K 8644 (100 nM) and FPL 64176 (150 nM), augmented by 3 microM thapsigargin plus 200 nM ryanodine and unaffected by KN-93 and KB-R7943. Phase II was accelerated by the Ca(2+) channel agonists and by isoproterenol but was eliminated by thapsigargin plus ryanodine. The results suggest that phase I of electromechanical restitution is caused by a transient L-type Ca(2+) current facilitation, whereas phase II represents the recovery of the ability of the sarcoplasmic reticulum to release Ca(2+).

Mechanical alternans and restitution in failing SHHF rat left ventricles.

We examined mechanical alternans and electromechanical restitution in normal and failing rat hearts. Alternans occurred at 5 Hz in failing versus 9 Hz in control hearts and was reversed by 300 nM isoproterenol, 6 mM extracellular Ca(2+), 300 nM -BAY K 8644, or 50 nM ryanodine. Restitution curves comprised phase I, which was completed before relaxation of the steady-state beat, and phase II, which occurred later. Phase I action potential area and developed pressure ratios were significantly reduced in the failing versus control hearts. Phase II was a monoexponential increase in relative developed pressure as the extrasystolic interval was increased. The plateau of phase II was significantly elevated in failing hearts. Thapsigargin (3 microM) plus ryanodine (200 nM) potentiated phase I to a significantly greater extent in control versus failing hearts and abolished phase II in both groups. The results suggest that both regulation of Ca(2+) influx across the sarcolemma and Ca(2+) release by the sarcoplasmic reticulum may contribute to altered excitation-contraction coupling in the failing spontaneously hypertensive heart failure prone rat heart.