Cardiac overexpression of alcohol dehydrogenase exacerbates cardiac contractile dysfunction,lipid peroxidation,and protein damage after chronic ethanol ingestion

BACKGROUND: Alcoholic cardiomyopathy is manifested as ventricular dysfunction, although its specific toxic mechanism remains obscure. This study was designed to examine the impact of enhanced acetaldehyde exposure on cardiac function via cardiac-specific overexpression of alcohol dehydrogenase (ADH) after alcohol intake. METHODS: ADH transgenic and wild-type FVB mice were placed on a 4% alcohol or control diet for 8 weeks. Mechanical and intracellular Ca2+ properties were evaluated in cardiac myocytes. Levels of acetaldehyde, lipid peroxidation, and protein carbonyl formation were determined. RESULTS: FVB and ADH mice consuming ethanol exhibited elevated blood ethanol/acetaldehyde, cardiac acetaldehyde, and cardiac hypertrophy compared with non-ethanol-consuming mice. However, the levels of cardiac acetaldehyde and hypertrophy were significantly greater in ADH ethanol-fed mice than FVB ethanol-fed mice. ADH transgene itself did not affect mechanical and intracellular Ca2+ properties with the exception of reduced resting intracellular Ca2+ and Ca2+ re-sequestration at low pace frequency. Myocytes from ethanol-fed mice showed significantly depressed peak shortening, velocity of shortening/relengthening, rise of intracellular Ca2+ transients, and sarco(endo)plasmic reticulum Ca2+ load associated with similar duration of shortening/relengthening compared with myocytes from control mice. Strikingly, the ethanol-induced mechanical and intracellular Ca2+ defects were exacerbated in ADH myocytes compared with the FVB group except velocity of shortening/relengthening. The lipid peroxidation end products malondialdehyde and protein carbonyl formation were significantly elevated in both livers and hearts after chronic ethanol consumption, with the cardiac lipid and protein damage being exaggerated by ADH transgene. CONCLUSION: These data suggest that increased cardiac acetaldehyde exposure due to ADH transgene may play an important role in cardiac contractile dysfunctions associated with lipid and protein damage after alcohol intake.     

Cardiac overexpression of metallothionein attenuates chronic alcohol intake-induced cardiomyocyte contractile dysfunction

Chronic alcohol ingestion leads to alcoholic cardiomyopathy manifested by ventricular dysfunction and heart failure. Although accumulation of reactive oxygen species may play a role in alcohol-induced heart injury, direct impact of enhanced antioxidant defense on pathogenesis of alcoholic cardiomyopathy has not been elucidated. This study was designed to examine the effect of transgenic overexpression of the free radical scavengermetallothionein on alcohol-induced cardiac contractile dysfunction. Wild-type FVB and metallothionein mice were placed on a 4% alcohol or control diet for 12 wk. Cardiac contractile function was evaluated in cardiomyocytes including peak shortening (PS), time-to-peak shortening, time-to-90% relengthening (TR₉₀), maximal velocity of shortening/relengthening (±dL/dt), intracellular Ca²⁺ rise (change in fura-2 fluorescent intensity [ΔFF1]) and intracellular Ca²⁺ decay rate. Intracellular Ca²⁺ cycling proteins including sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), Na⁺−Ca²⁺ exchanger (NCX) and phospholamban were assessed using Western blot analysis. Alcohol intake depressed PS, ±dL/dt, and ΔFF1, increased baseline fura-2 fluorescence intensity (FF1), and prolonged intracellular Ca²⁺ decay and TR₉₀, all of which with the exception of ΔFF1 were abrogated by metallothionein. Enhanced stimulating frequency caused lessened PS decline at 1.0 Hz from FVB ethanol group, which was not affected by metallothionein. Immunoblotting data showed reduced SERCA2a, NCX and phospholamban expression in FVB group consuming alcohol. All of these alcohol-induced changes in cardiac proteins were nullified by the metallothionein transgene. In summary, our findings suggest a beneficial role of antioxidants in alcohol-induced cardiomyocyte dysfunction.     

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).     

Effects of Na(+)/Ca(2+)-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.     

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.     

A SERCA2 pump with an increased Ca2+ affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span

Abnormal Ca(2+) cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca(2+) pump, the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca(2+) by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca(2+) affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB(-/-) mice. The affinity for Ca(2+) of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca(2+)-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca(2+) is physiologically important for normal cardiac function. An improved Ca(2+) transport in the sarcoplasmic reticulum may correct Ca(2+) mishandling in heart failure, but a SERCA pump with a much higher Ca(2+) affinity may be detrimental.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Influence of gender on intrinsic contractile properties of isolated ventricular myocytes from calmodulin-induced diabetic transgenic mice

Diabetes mellitus impairs ventricular function, which itself may be disparately influenced by gender. This study compared the impact of gender on cardiac contractile response in ventricular myocytes from wild-type FVB and calmodulin-induced diabetic transgenic (OVE26) mice at young (2 month) and older (11 month) age. Mechanical and intracellular Ca2+ properties of cardiac myocytes were evaluated using an IonOptix MyoCam system. Diabetic mice of both genders exhibited significantly elevated blood glucose regardless of age. OVE26 myocytes displayed reduced peak shortening (PS) and maximal velocity of shortening/relengthening (+/- dL/dt), and prolonged time-to-PS (TPS) and time-to-90% relengthening (TR90), associated with higher resting intracellular Ca2+ levels and attenuated Ca(2+)-induced intracellular Ca2+ release compared with the FVB myocytes. Peak shortening and +/- dL/dt were smaller in female FVB groups when compared to the age-matched male counterparts. However, these gender differences were ablated by the diabetic state. No significant gender-related differences in intracellular Ca2+ handling were noted in either FVB or OVE26 myocytes, with the exception of overt gender differences in OVE26 mice when age was taken into account. Young female OVE26 mice exhibited better-preserved mechanical function while older female OVE26 mice displayed the worst mechanical function among all four OVE26 groups. In conclusion, our data confirmed impaired cardiac contractile function in diabetes, partially due to altered intracellular Ca2+ handling, in both genders. Mechanical differences existed between genders but were "cancelled off" by diabetic state. Nevertheless, a "female advantage" in ventricular function may still persist in young female diabetic subjects.     

Inhibition of cardiac myocyte contraction by 4-hydroxy-trans-2-nonenal

Lipid peroxidation, initiated by hydroxyl radicals, results in production of 4-Hydroxy-trans-2-nonenal (HNE) and leads to cardiac injury. However, impact of HNE on ventricular function has not been clearly defined. This study was to examine the direct effect of HNE on cardiac contractile function at cardiac myocyte level. Adult male rat ventricular myocytes were isolated and electrically stimulated to contract at 0.5 Hz. Mechanical and intracellular Ca2+ properties were evaluated using an Ionoptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-PS, time-to-90% relengthening, maximal velocities of shortening and relengthening (+/-dL/dt), change of electrically stimulated intracellular Ca2+ fura-2 fluorescent intensity, and intracellular Ca2+ decay. Our results indicated that short-term incubation of HNE (10(-6) to 10(-4) M) with myocytes depressed PS, +/-dL/dt, and fura-2 fluorescent intensity; shortened time-PS; and elevated resting intracellular Ca2+ levels without affecting time-to-90% relengthening and intracellular Ca2+ decay. Interestingly, the HNE-induced cardiac mechanical effects (with the exception of shortened time-PS) were abolished by either the aldehyde dehydrogenase inhibitor cyanamide or the p38 mitogen-activated protein kinase inhibitor SB203580. These findings reveal a possible role of HNE in the lipid peroxidation-associated cardiac contractile dysfunction that is likely mediated through HNE metabolism and mitogen-activated protein kinase activation.     

Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size

Spontaneous, localized, rhythmic ryanodine receptor (RyRs) Ca(2+) releases occur beneath the cell membrane during late diastolic depolarization in cardiac sinoatrial nodal cells (SANCs). These activate the Na(+)/Ca(2+) exchanger (NCX1) to generate inward current and membrane excitation that drives normal spontaneous beating. The morphological background for the proposed functional of RyR and NCX crosstalk, however, has not been demonstrated. Here we show that the average isolated SANC whole cell labeling density of RyRs and SERCA2 is similar to atrial and ventricle myocytes, and is similar among SANCs of all sizes. Labeling of NCX1 is also similar among SANCs of all sizes and exceeds that in atrial and ventricle myocytes. Submembrane colocalization of NCX1 and cardiac RyR (cRyR) in all SANCs exceeds that in the other cell types. Further, the Cx43 negative primary pacemaker area of the intact rabbit sinoatrial node (SAN) exhibits robust positive labeling for cRyR, NCX1, and SERCA2. Functional studies in isolated SANCs show that neither the average action potential (AP) characteristics, nor those of intracellular Ca(2+) releases, nor the spontaneous cycle length vary with cell size. Chelation of intracellular [Ca(2+)], or disabling RyRs or NCX1, markedly attenuates or abolishes spontaneous SANC beating in all SANCs. Thus, there is dense labeling of SERCA2, RyRs, and NCX1 in small-sized SANCs, thought to reside within the SAN center, the site of impulse initiation. Because normal automaticity of these cells requires intact Ca(2+) cycling, interactions of SERCA, RyR2 and NCX molecules are implicated in the initiation of the SAN impulse.     

Influence of interleukin-2 on Ca2+ handling in rat ventricular myocytes

In the present study, we examined the effect of interleukin-2 (IL-2) on cardiomyocyte Ca(2+) handling. The effects of steady-state and transient changes in stimulation frequency on the intracellular Ca(2+) transient were investigated in isolated ventricular myocytes by spectrofluorometry. In the steady state (0.2 Hz) IL-2 (200 U/ml) decreased the amplitude of Ca(2+) transients induced by electrical stimulation and caffeine. At 1.25 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), when the stimulation frequency increased from 0.2 to 1.0 Hz, diastolic Ca(2+) level and peak intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the amplitude of the transient, increased. The positive frequency relationships of the peak and amplitude of [Ca(2+)](i) transients were blunted in the IL-2-treated myocytes. The effect of IL-2 on the electrically induced [Ca(2+)](i) transient was not normalized by increasing [Ca(2+)](o) to 2.5 mM. IL-2 inhibited the frequency relationship of caffeine-induced Ca(2+) release. Blockade of sarcoplasmic reticulum (SR) Ca(2+)-ATPase with thapsigargin resulted in a significant reduction of the amplitude-frequency relationship of the transient similar to that induced by IL-2. The restitutions were not different between control and IL-2 groups at 1.25 mM [Ca(2+)](o), which was slowed in IL-2-treated myocytes when [Ca(2+)](o) was increased to 2.5 mM. There was no difference in the recirculation fraction (RF) between control and IL-2-treated myocytes at both 1.25 and 2.5 mM [Ca(2+)](o). The effects of IL-2 on frequency relationship, restitution, and RF may be due to depressed SR functions and an increased Na(+)-Ca(2+) exchange activity, but not to any change in L-type Ca(2+) channels.     

Transgenic overexpression of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile dysfunction and beta-adrenergic response in ventricular myocytes

Diabetic cardiomyopathy is characterized by cardiac dysfunction and altered level/function of insulin-like growth factor I (IGF-I). Both endogenous and exogenous IGF-I have been shown to effectively alleviate diabetes-induced cardiac dysfunction and oxidative stress. This study was designed to examine the effect of cardiac overexpression of IGF-I on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse myocytes. Both IGF-I heterozygous transgenic mice and their wild-type FVB littermates were made diabetic with a single injection of STZ (200 mg/kg, i.p.) and maintained for 2 weeks. The following mechanical indices were evaluated in ventricular myocytes: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Intracellular Ca2+ was evaluated as resting and peak intracellular Ca2+ levels, Ca2+-induced Ca2+ release and intracellular Ca2+ decay rate (tau). STZ led to hyperglycemia in FVB and IGF-I mice. STZ treatment prolonged TPS and TR90, reduced Ca2+-induced Ca2+ release, increased resting intracellular Ca2+ levels and slowed tau associated with normal PS and +/- dL/dt. All of which, except the elevated resting intracellular Ca2+, were prevented by the IGF-I transgene. In addition, myocytes from STZ-treated FVB mice displayed an attenuated contractile response to the beta-adrenergic agonist isoproterenol, which was restored by the IGF-I transgene. Contractile response to the alpha-adrenergic agonist phenylephrine and angiotensin II was not affected by either STZ treatment or IGF-I. These results validate the beneficial role of IGF-I in diabetic cardiomyopathy, possibly due to an improved beta-adrenergic response.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy

We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca(2+) removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca(2+) removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca(2+) transients were prolonged in HG cells (A(T/PK)=215+/-7ms, n=41) compared to N myocytes (A(T/PK)=173+/-5ms, n=34). By pharmacological and ionic manipulations, Ca(2+) removal attributable to SERCA was slower in the HG group (A(D/PK)=290+/-17ms,n =41) compared to N (A(D/PK)=219+/-10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. beta-adrenergic receptor stimulation with ISO (10(-7)M) normalized myocyte relaxation, Ca(2+) transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10(-5)M) depressed peak fractional shortening (PS) and slowed relengthening (A(R/PK)) to a greater extent in N (-50% for PS and 92% for A(R/PK)) than in HG cells (-25% for PS and 48% A(R/PK)). Depressed cytosolic Ca(2+) removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca(2+) regulation, which may be a common manifestation of other forms of cardiomyopathies.     

Insulin-like growth factor I deficiency prolongs survival and antagonizes paraquat-induced cardiomyocyte dysfunction: role of oxidative stress

Interruption of insulin-like growth factor I (IGF-1) signaling has been demonstrated to prolong life span although the underlying mechanism has not been elucidated. The aim of this study was to examine the influence of severe IGF-1 deficiency on survival rate, cardiomyocyte viability, contractile function, and intracellular Ca(2+) property in response to challenge with the pro-oxidant paraquat. C57 negative and liver IGF-1 deficient (LID) transgenic mice were administrated paraquat (75 mg/kg) and survival was monitored. LID mice displayed a significantly improved survival than did C57 mice evaluated by the Kaplan-Meier curve. MTT assay revealed that in vitro IGF-1 treatment significantly sensitized paraquat-induced cell death in both C57 and LID groups, with significantly better cell viability in LID cardiomyocytes. Compared to C57 mouse cardiomyocytes, LID myocytes displayed reduced peak shortening (PS), decreased maximal velocity of shortening/relengthening (+/- dL/dt), prolonged time-to-90% relengthening (TR(90)), and comparable tolerance to high stimulus frequency and intracellular Ca(2+) homeostasis. Paraquat treatment for 48 hours reduced PS, +/- dL/dt, tolerance to high stimulus frequency, resting and rise in intracellular Ca(2+), and prolonged TR(90), all of which were nullified or masked by IGF-1 deficiency. Paraquat increased reactive oxygen species and carbonyl production upregulated the Ca(2+) regulating protein SERCA2a, and downregulated Na(+) -Ca(2+) exchanger, the effects of which were nullified or masked by IGF-1 deficiency. Although LID mice displayed reduced whole body glucose clearance, cardiomyocytes from LID mice exhibited dramatically enhanced insulin-stimulated phosphorylation of insulin receptor and Akt. These data demonstrated that IGF-1 deficiency may antagonize or mask the paraquat-induced decrease in survival, cardiomyocyte dysfunction, oxidative stress, and change in Ca(2+) regulating proteins.     

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+–Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca²⁺ homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca²⁺ fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca²⁺-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca²⁺ release channels (RR), and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca²⁺ uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% ± 1% vs 64% ± 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% ± 1% vs 34.1% ± 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (–dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca²⁺ uptake (nmol ⁴⁵Ca²⁺ accumulated/min per milligram noncollagen protein) declined by 25% (21.3 ± 1 vs 28.5 ± 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca²⁺ uptake, Ca²⁺ release, and Na⁺–Ca²⁺ exchange are critically associated with the deterioration of LV function during the progression of HF.     

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.     

Impaired cardiac contractile function in ventricular myocytes from leptin-deficient ob/ob obese mice

The level of the obese gene product leptin is often positively correlated with body weight, supporting the notion that hyperleptinemia contributes to obesity-associated cardiac dysfunction. However, a link between leptin levels and cardiac function has not been elucidated. This study was designed to examine the role of leptin deficiency (resulting from a point mutation of the leptin gene) in cardiomyocyte contractile function. Mechanical properties and intracellular Ca(2+) transients were evaluated in ventricular myocytes from lean control and leptin-deficient ob/ob obese mice at 12 weeks of age. Cardiac ultrastructure was evaluated using transmission electron microscopy. ob/ob mice were overtly obese, hyperinsulinemic, hypertriglycemic, hypoleptinemic and euglycemic. Ultrastructural examination revealed swelling and disorganization of cristae in mitochondria from ob/ob mouse ventricular tissues. Cardiomyocytes from ob/ob mice displayed reduced expression of the leptin receptor Ob-R, larger cross-sectional area, decreased peak shortening and maximal velocity of shortening/relengthening, and prolonged relengthening but not shortening duration compared with lean counterparts. Consistent with mechanical characteristics, myocytes from ob/ob mice displayed reduced intracellular Ca(2+) release upon electrical stimulus associated with a slowed intracellular Ca(2+) decay rate. Interestingly, the contractile aberrations seen in ob/ob myocytes were significantly improved by in vitro leptin incubation. Contractile dysfunction was not seen in age- and gender-matched high fat-induced obese mice. These results suggested that leptin deficiency contributes to cardiac contractile dysfunction characterized by both systolic and diastolic dysfunction, impaired intracellular Ca(2+) hemostasis and ultrastructural derangement in ventricular myocytes.