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Background Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs. Methods Cultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 μg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, and the expression of phosphorylated CaMKIIδ and NOX2/gp91phox. The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique. Results In NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP. Conclusions L5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease.

Background Oxidized low-density lipoprotein (ox-LDL) is crucial in cardiac injury. Apolipoprotein-J (ApoJ) contributes to antiapoptotic effects in the heart. We aimed to evaluate the protective effects of ApoJ against ox-LDL cytotoxicity in Neonatal rat ventricular cells (NRVCs). Methods and results NRVCs were damaged by exposure to ox-LDL, as shown by increased caspase-3/7 activity, enhanced caspase-3 expression, and decreased cell viability. ApoJ overexpression, using an adenovirus vector, significantly reduced ox-LDL-induced cell injury. ApoJ also prevented ox-LDL from augmenting reactive oxygen species (ROS) production, as demonstrated by elevated Nox2/gp91phox and P47 expression. Furthermore, ApoJ overexpression reduced CaMKIIδ expression elicited by ox-LDL in cultured NRVCs. Upregulating CaMKIIδ activity, mediated by ox-LDL, was significantly inhibited by ApoJ overexpression. A CaMKIIδ inhibitor, KN93, prevented ApoJ’s protective effect against ox-LDL cytotoxicity. A ROS scavenger, Mn (III)meso-tetrakis (4-benzoic acid) porphyrin (Mn (III)TBAP), also attenuated CaMKIIδ’s increased expression and activity, induced by ox-LDL, and showed similar results to ApoJ by attenuating ox-LDL-induced cell damage, as ApoJ did. Conclusions ApoJ confers cytoprotection to NRVCs against ox-LDL cytotoxicity through the ROS-CaMKII pathways.

Thel-type Ca(2+)current (ICa,l) plays a crucial role in shaping action potential and is involved in cardiac arrhythmia. Statins have been demonstrated to contribute to anti-apoptotic and anti-arrhythmic effects in the heart. Here, we examined whether atorvastatin regulates theICa,land cell injury induced by angiotensin II (AngII) as well as the putative intracellular cascade responsible for the effects. Cultured neonatal rat ventricular myocytes were incubated with AngII for 24 h, and then cell injury and expression levels of Nox2/gp91(phox), p47(phox) ,and Cav1.2 were analyzed. In addition,ICa,lwas recorded using the whole-cell patch-clamp technique, and mechanisms of atorvastatin actions were also investigated. It was found that the number of apoptotic cardiomyocytes was increased and cell viability was significantly decreased after AngII administration. AngII also augmented the expressions of Nox2/gp91(phox)and p47(phox)compared with control cardiomyocytes. Exposure to AngII evokedICa,lin a voltage-dependent manner without affecting theI-Vrelationship. In addition, AngII enhanced membrane Cav1.2 expression. These effects were abolished in the presence of the reactive oxygen species (ROS) scavenger, manganese (III)-tetrakis 4-benzoic acid porphyrin [Mn(III)TBAP], or the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, atorvastatin. These results suggested that atorvastatin mediates cardioprotection against arrhythmias and cell injury by controlling the AngII-ROS cascade.

BACKGROUND This study investigated effects of exhaustive exercise on L-type calcium current (ICa,L) and the putative intracellular cascade responsible for the effects. METHODS Rats were randomly divided into three treatment groups: sedentary (without exercise), exercised to exhaustion and salubrinal injection before each exhaustive exercise period. Exercise group rats were forced to swim until exhaustion each time for 9 days with 5% body weight attached to the head. Salubrinal (1 mg/kg) or an equivalent volume of placebo solution (dimethyl sulfoxide) was injected via the intraperitoneal route daily for the first 3 days, followed by subcutaneous injections of salubrinal (0.5 mg/kg) or placebo solution daily for 9 days (starting 30 min before exercise). After a 1-day recovery period, whole-cell patch clamping was used to investigate the L-type Ca2+ current (ICa,L), with sedentary control rats. Additionally, endoplasmic reticulum (ER) chaperone protein levels were analyzed. RESULTS Exhaustive exercise triggered ER stress, demonstrated by elevated expression of ER stress markers: phospho-eIF2α, CCAT/enhancer-binding homologous protein (CHOP) and caspase-12. Compared to controls, ICa,L was inhibited by exhaustive exercise, which was blocked by salubrinal, a selective eIF2α dephosphorylation inhibitor used to inhibit ER stress. These results suggest that ER stress participates in regulation of ICa,L. However, exhaustive exercise did not change the voltage dependence of steady-state activation and inactivation of ICaL, and salubrinal infusion caused no difference in voltage dependence of steady-state activation and inactivation of ICa,L. CONCLUSIONS Exhaustive exercise activates ER stress, thus inhibiting ICaL, which may change the action potential duration and contribute to proarrhythmia.

Background Up-regulation of angiotensin II (AngII) occurs in cardiac diseases, such as congestive heart failure, cardiac hypertrophy, myocardial ischemia and atrial fibrillation, which represent major health problems. Evidence from in vivo studies suggests that the level of Apolipoprotein-J (ApoJ) is also elevated but plays a protective role in cardiovascular disease. This study aimed to evaluate the protective effects of ApoJ against cytotoxicity of AngII in neonatal rat ventricular cells (NRVCs). Methods and results In culture, NRVCs were damaged by exposure to AngII, and ApoJ overexpression using an adenovirus vector significantly reduced the AngII-induced cell injury. ApoJ also prevented AngII from augmenting Nox2/gp91phox expression. The reactive oxygen species (ROS) scavenger, Mn(III)TBAP, showed similar results of attenuating AngII-induced cell damage. Furthermore, ApoJ overexpression increased phosphorylation of Akt, and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 diminished the antioxidant effects of ApoJ, and prevented the protective effect of ApoJ against the cytotoxicity of AngII. Moreover, upregulation of nuclear factor κB (NF-κB) p65 expression and phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated by AngII in cultured NRVCs were significantly inhibited by overexpression of ApoJ. The p38 MAPK inhibitor SB203580 and the NF-κB inhibitor PDTC protected NRVCs from injury caused by AngII. Conclusions ApoJ serves as a cytoprotective protein in NRVCs against cytotoxicity of AngII through the PI3K-Akt-ROS and MAPK/ NF-κB pathways.

The receptor for advanced glycation end products (RAGE) and thioredoxin (Trx) play opposing roles in diabetic myocardial ischemia-reperfusion (MI/R) injury. We recently demonstrated nitrative modification of Trx leads to its inactivation and loss of cardioprotection. The present study is to determine the relationship between augmented RAGE expression and diminished Trx activity pertaining to exacerbated MI/R injury in the diabetic heart. The diabetic state was induced in mice by multiple intraperitoneal low-dose streptozotocin injections. RAGE small-interfering RNA (siRNA) or soluble RAGE (sRAGE, a RAGE decoy) was via intramyocardial and intraperitoneal injection before MI/R, respectively. Mice were subjected to 30 min of myocardial infarction followed by 3 or 24 h of reperfusion. At 10 min before reperfusion, diabetic mice were randomized to receive EUK134 (peroxynitrite scavenger), recombinant hTrx-1, nitrated Trx-1, apocynin (a NADPH oxidase inhibitor), or 1400W [an inducible nitric oxide synthase (iNOS) inhibitor] administration. The diabetic heart manifested increased RAGE expression and Nε-(carboxymethyl)lysine (CML, major advanced glycation end product subtype) content, reduced Trx-1 activity, and increased Trx nitration after MI/R. RAGE siRNA or administration of sRAGE in diabetic mice decreased MI/R-induced iNOS and gp91phox expression, reduced Trx nitration, preserved Trx activity, and decreased infarct size. Apocynin or 1400W significantly decreased nitrotyrosine production and restored Trx activity. Conversely, administration of either EUK134 or reduced hTrx, but not nitrated hTrx, attenuated MI/R-induced superoxide production, RAGE expression, and CML content and decreased cardiomyocyte apoptosis in diabetic mice. Collectively, we demonstrate that RAGE modulates the MI/R injury in a Trx nitrative inactivation fashion. Conversely, nitrative modification of Trx blocked its inhibitory effect upon RAGE expression in the diabetic heart. This is the first direct evidence demonstrating the alternative cross talk between RAGE overexpression and nitrative Trx inactivation, suggesting that interventions interfering with their interaction may be novel means of mitigating diabetic MI/R injury.

Type 1 diabetes (T1DM) portends poor prognosis concerning ischemic heart disease. Adiponectin (APN), an adipocytokine possessing insulin sensitizing and metabolic regulatory effects, has been recognized as a potent cardioprotective molecule. However, the relationship between APN and T1DM remains controversial and the role of cardiac-derived APN in T1DM is unclear. This study is aimed to investigate the dynamic change of both plasma and cardiac-derived APN expressions in T1DM, and the particular role of cardiac-derived APN in T1DM against myocardial ischemia/reperfusion (MI/R) injury. T1DM was established via intraperitoneal injection of streptozocin and followed by twice-daily subcutaneous injection of insulin or vehicle for 14 days. Non-diabetic mice of wild type and APN knockout were subjected to insulin or vehicle injection. MI/R was induced in Langendorff-perfused hearts. Compared to non-diabetic mice, plasma APN levels of diabetic mice significantly increased at 7 days, and slightly decreased at 14 days, while cardiac-derived APN levels gradually decreased over time. The MI/R injury measured as infarct size and cardiomyocyte apoptosis nearly doubled in diabetic mice. 14 days of insulin treatment increased both plasma and cardiac-derived APN levels in diabetic mice and attenuated myocardial injury via increasing AMPK phosphorylation in T1DM, which was partly reversed by Compound C (an AMPK inhibitor). Moreover, APN deficiency aggravated MI/R injury and partly abolished the protective effect of insulin treatment against MI/R injury, which was associated with decreased AMPK phosphorylation. The results suggest that cardiac-derived APN stimulated by long-term insulin treatment in T1DM exerts cardioprotection against MI/R injury via myocardial AMPK activation.

Objective Adiponectin, circulates as trimer, hexamer and high molecular weight form (HMW) in blood. Both adiponectin complexes concentration and their bioactivities are decreased in pathological state. Moreover, DsbA-L, a key regulator for adiponectin biosynthesis, is down-regulated in obese humans. However, the alterations of adiponectin multimers distribution, bioactivities and DsbA-L level in newly diagnosed Chinese type 2 diabetes (T2DM) patients are unknown. The objective of present study was to compare plasma adiponectin isoform status along with their bioactivities and DsbA-L concentration in T2DM with control subjects. Methods Plasma total adiponectin was measured by ELISA. Adiponectin multimers were analysed by western blot followed SDS-PAGE under nonheating nonreducing conditions. Plasma adiponectin multimers were isolated by chromotography techniques from plasma in ten T2DM and ten healthy control subjects respectively. Adiponectin bioactivity was detected by incubating HUVEC with isolated adiponectin isoforms and determining the phosphorylation level of AMPK and eNOS. DsbA-L protein expression level was determined by western blot. Results 189 T2DM (48.7±9.4 y) and 123 healthy people (42.8±8.0 y) were enrolled. In comparison with healthy control group, total adiponectin (p Conclusion This study directly demonstrated for the first time that human HMW adiponectin bioactivity was decreased in type 2 diabetes. We also provide a target for regulating HMW adiponectin concentration in diabetic patients.