Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion. Am J Physiol Heart Circ Physiol 301: H1695-H1705, 2011. First published August 5, 2011; doi:10.1152/ajpheart.00276.2011.-Intermittent hypobaric hypoxia (IHH) protects hearts against ischemiareperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 Ϯ 5.3% in IHH vs. 42.1 Ϯ 3.8% in the normoxic control, P Ͻ 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K ϩ channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3␤, as well as translocation of PKC-ε were not affected by applying H 2O2 (20 mol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl-)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHHinduced cardioprotection depends on elevated ROS production during early reperfusion.reactive oxygen species; ischemia-reperfusion injury EARLY REPERFUSION during evolving myocardial infarction is essential for saving the myocardium, but lethal reperfusion injury can occur and limit the beneficial effects (49). A number of cardioprotective strategies have been developed to ameliorate or retard the irreversible injury. However, the clinical translation of these strategies has failed to achieve the anticipated results (13, 34). Intermittent hypobaric hypoxia (IHH) has been shown to protect the heart against ischemia-reperfusion (I/R) injury by improving the manifestations including contractile dysfunction (3, 33), arrhythmias (31, 52), and cell death (8,27). Recently, we (48) revealed a therapeutic effect of IHH on permanent coronary artery ligation-induced myocardial infarction by attenuating infarct size, myocardial fibrosis, and apoptosis and improving cardiac performance. Because IHH is a relatively simple intervention with a longer protection duration and fewer adverse effects and may offer profound benefit to patients ...

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

E2F6 is believed to repress E2F-responsive genes and therefore plays an important role in cell-cycle regulation. However, the role of E2F6 in the control of apoptosis remains unknown. We show here that the expression of E2F6 was downregulated with a concurrent increase in BRCA1 mRNA and cleaved protein during ultraviolet (UV)-induced apoptosis in human embryonic kidney 293 cells. Moreover, E2F6 overexpression distinctly inhibited UV-induced apoptosis as well as UV-induced increases in BRCA1 expression and cleavage, accompanied with increases of the full-length BRCA1 and BRCA1 nuclear foci. In contrast, knockdown of E2F6 by small interfering RNA had opposite effects. Furthermore, these effects of E2F6 on BRCA1 depended upon the association of E2F6 with BRCA1 via its C-terminus in a UV-triggered manner and upon the transcriptional repression by E2F6 on the BRCA1 promoter. These findings provide the first demonstration of the important role for E2F6 in the control of apoptosis via targeting of BRCA1.

TMBIM1 protects against pathological cardiac hypertrophy through promoting the lysosomal degradation of activated TLR4. Our findings reveal the central role of TMBIM1 as a multivesicular body regulator in the progression of pathological cardiac hypertrophy, as well as the role of vesicle trafficking in signaling regulation during cardiac hypertrophy. Moreover, targeting TMBIM1 could be a novel therapeutic strategy for treating cardiac hypertrophy and heart failure.

Reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are paradoxically implicated in myocardial ischemia/reperfusion (I/R) injury and cardioprotection. However, the precise interpretation for the dual roles of ROS and its relationship with the ER stress during I/R remain elusive. Here we investigated the concentration-dependent effects of hydrogen peroxide (H2O2) preconditioning (PC) and postconditioning (PoC) on the ER stress and prosurvival reperfusion injury salvage kinase (RISK) activation using an ex vivo rat myocardial I/R model. The effects of H2O2 PC and PoC showed three phases. At a low level (1 μM), H2O2 exacerbated I/R-induced left ventricular (LV) contractile dysfunction and ER stress, as indicated by enhanced phosphorylation of protein kinase-like ER kinase and expressions of glucose-regulated protein 78, X-box-binding protein 1 splicing variant, TNF receptor-associated factor 2, activating transcription factor-6 cleaved 50 kDa fragment, and caspase-12 cleavage, but the I/R-induced RISK activation including protein kinase B (PKB/Akt) and protein kinase Cɛ (PKCɛ) remained unchanged. Consistently, the postischemic LV performance in 1 μM H2O2 PC and PoC groups was improved by inhibiting ER stress with 4-phenyl butyric acid but not affected by the ER stress inducer, tunicamycin. At a moderate level (10–100 μM), H2O2 significantly improved postischemic LV performance and enhanced RISK activation, but it did no further alter the ER stress. The cardioprotection but not ER stress was abrogated with Akt or PKCɛ inhibitor wortmannin or ɛV1–2. At a high level (1 mM), H2O2 markedly aggravated the reperfusion injury and the oxidative stress but did not further enhance the RISK activation. In addition, 1 or 20 μM of H2O2 PC did not alter cardioprotective effects of ischemic PC in postischemic contractile performance and protein oxidation. Our data suggest that the differential effects of H2O2 are derived from a concentration-dependent wrestling between its detrimental stress and protective signaling.

Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either -knockout or-overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3β (glycogen synthase kinase-3β)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by deficiency but suppressed by overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy.

Background: MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through canonical RNA interference (RNAi) mechanism. However, it remains unknown if endogenous miRs modulate the physiological homeostasis of the heart through noncanonical mechanisms. Methods: We focused on the predominant miR of the heart--miR1 and investigated if miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay (EMSA), in situ proximity ligation assay (PLA), RNA pull down and RNA Immunoprecipitation (RIP) assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was utilized to understand the underlying mechanism. The effect on the ex vivo heart was demonstrated through investigating arrhythmia-associated human single nucleotide-polymorphisms (hSNPs) with miR1-deficient mice. Results: We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine and human cardiomyocytes. miR1 quickly and significantly suppressed I K1 at sub-pmol/L concentration, which is close to endogenous miR-expression level. Acute presence of miR1 depolarized resting membrane potential (RMP) and prolonged final repolarization of the action potential in cardiomyocytes. We identified three miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNAi seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short-QT/AF patients. We found that an arrhythmia-associated hSNP of miR1--hSNP14A/G specifically disrupts the biophysical modulation while retaining the RNAi function. Remarkably, miR1 but not hSNP14A/G relieved the hyperpolarized RMP in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo . Conclusions: Our study reveals a novel evolutionarily-conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion-channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

Intermittent high-altitude (IHA) hypoxia-induced cardioprotection against ischemia-reperfusion (I/R) injury is associated with the preservation of sarcoplasmic reticulum (SR) function. Although Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and phosphatase are known to modulate the function of cardiac SR under physiological conditions, the status of SR CaMKII and phosphatase during I/R in the hearts from IHA hypoxic rats is unknown. In the present study, we determined SR and cytosolic CaMKII activity during preischemia and I/R (30 min/30 min) in perfused hearts from normoxic and IHA hypoxic rats. The left ventricular contractile recovery, SR CaMKII activity as well as phosphorylation of phospholamban at Thr(17), and Ca(2+)/CaM-dependent SR Ca(2+)-uptake activity were depressed in the I/R hearts from normoxic rats, whereas these changes were prevented in the hearts from IHA hypoxic rats. Such beneficial effects of IHA hypoxia were lost by treating the hearts with a specific CaMKII inhibitor, KN-93. I/R also depressed cytosolic CaMKII and SR phosphatase activity, but these alterations remained unchanged in IHA hypoxic group. Furthermore, we found that the autophosphorylation at Thr(287), which confers Ca(2+)/CaM-independent activity, was not altered by I/R in both groups. These findings indicate that preservation of SR CaMKII activity plays an important role in the IHA hypoxia-induced cardioprotection against I/R injury via maintaining SR Ca(2+)-uptake activity.