Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.
Sudden unexpected cardiac death (SCD) accounts for up to half of all-cause mortality of heart failure patients. Standardized cardiology tools such as electrocardiography, cardiac imaging, electrophysiological and serum biomarkers cannot accurately predict which patients are at risk of life-threatening arrhythmic episodes. Recently, a common variant of the histidine-rich calcium binding protein (HRC), the Ser96Ala, was identified as a potent biomarker of malignant arrhythmia triggering in these patients. HRC has been shown to be involved in the regulation of cardiac sarcoplasmic reticulum (SR) Ca(2+) cycling, by binding and storing Ca(2+) in the SR, as well as interacting with the SR Ca(2+) uptake and release complexes. The underlying mechanisms, elucidated by studies at the molecular, biochemical, cellular and intact animal levels, indicate that transversion of Ser96 to Ala results in abolishment of an HRC phosphorylation site by Fam20C kinase and dysregulation of SR Ca(2+) cycling. This is mediated through aberrant SR Ca(2+) release by the ryanodine receptor (RyR2) quaternary complex, due to the impaired HRC/triadin interaction, and depressed SR Ca(2+) uptake by the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2) pump, due to the impaired HRC/SERCA2 interaction. Pharmacological intervention with KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), in the HRC Ser96Ala mouse model, reduced the occurrence of malignant cardiac arrhythmias. Herein, we summarize the current evidence on the pivotal role of HRC in the regulation of cardiac rhythmicity and the importance of HRC Ser96Ala as a genetic modifier for arrhythmias in the setting of heart failure.
Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.
Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1(-/-) x Apoe(-/-) mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.
BACKGROUND: High Density Lipoprotein (HDL) and its main protein component, apolipoprotein A-I (apoA-I), have numerous atheroprotective functions on various tissues including the endothelium. Therapies based on reconstituted HDL containing apoA-I (rHDL-apoA-I) have been used successfully in patients with acute coronary syndrome, peripheral vascular disease or diabetes but very little is known about the genomic effects of rHDL-apoA-I and how they could contribute to atheroprotection. OBJECTIVE: The present study aimed to understand the endothelial signaling pathways and the genes that may contribute to rHDL-apoA-I-mediated atheroprotection. METHODS: Human aortic endothelial cells (HAECs) were treated with rHDL-apoA-I and their total RNA was analyzed with whole genome microarrays. Validation of microarray data was performed using multiplex RT-qPCR. The expression of ANGPTL4 in EA.hy926 endothelial cells was determined by RT-qPCR and Western blotting. The contribution of signaling kinases and transcription factors in ANGPTL4 gene regulation by HDL-apoA-I was assessed by RT-qPCR, Western blotting and immunofluorescence using chemical inhibitors or siRNA-mediated gene silencing. RESULTS: It was found that 410 transcripts were significantly changed in the presence of rHDL-apoA-I and that angiopoietin like 4 (ANGPTL4) was one of the most upregulated and biologically relevant molecules. In validation experiments rHDL-apoA-I, as well as natural HDL from human healthy donors or from transgenic mice overexpressing human apoA-I (TgHDL-apoA-I), increased ANGPTL4 mRNA and protein levels. ANGPTL4 gene induction by HDL was direct and was blocked in the presence of inhibitors for the AKT or the p38 MAP kinases. TgHDL-apoA-I caused phosphorylation of the transcription factor forkhead box O1 (FOXO1) and its translocation from the nucleus to the cytoplasm. Importantly, a FOXO1 inhibitor or a FOXO1-specific siRNA enhanced ANGPTL4 expression, whereas administration of TgHDL-apoA-I in the presence of the FOXO1 inhibitor or the FOXO1-specific siRNA did not induce further ANGPTL4 expression. These data suggest that FOXO1 functions as an inhibitor of ANGPTL4, while HDL-apoA-I blocks FOXO1 activity and induces ANGPTL4 through the activation of AKT. CONCLUSION: Our data provide novel insights into the global molecular effects of HDL-apoA-I on endothelial cells and identify ANGPTL4 as a putative mediator of the atheroprotective functions of HDL-apoA-I on the artery wall, with notable therapeutic potential.
HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6(S10F)) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6(S10F) with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6(S10F)-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.
The Mesio-Temporal Lobe Epilepsy syndrome is the most common form of intractable epilepsy. It is characterized by recurrence of focal seizures and is often associated with hippocampal sclerosis and drug resistance. We aimed to characterize the molecular changes occurring during the initial stages of epileptogenesis in search of new therapeutic targets for Mesio-Temporal Lobe Epilepsy. We used a mouse model obtained by intra-hippocampal microinjection of kainate and performed hippocampal whole genome expression analysis at 6h, 12h and 24h post-injection, followed by multilevel bioinformatics analysis. We report significant changes in immune and inflammatory responses, neuronal network reorganization processes and glial functions, predominantly initiated during status epilepticus at 12h and persistent after the end of status epilepticus at 24h post-kainate. Upstream regulator analysis highlighted Cyba, Cybb and Vim as central regulators of multiple overexpressed genes implicated in glial responses at 24h. In silico microRNA analysis indicated that miR-9, miR-19b, miR-129, and miR-223 may regulate the expression of glial-associated genes at 24h. Our data support the hypothesis that glial-mediated inflammatory response holds a key role during epileptogenesis, and that microglial cells may participate in the initial process of epileptogenesis through increased ROS production via the NOX complex.
BACKGROUND: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. OBJECTIVE: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. METHODS/RESULTS: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AbetaPP overexpressing SH-SY5Y-AbetaPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AbetaPP and tau processing pathways were investigated in the SH-SY5Y-AbetaPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3beta, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AbetaPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. CONCLUSIONS: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.
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