Guan-Sheng Liu

Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia

AIMS Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. METHODS AND RESULTS Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. CONCLUSION The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers.

HAX-1 regulates cyclophilin-D levels and mitochondria permeability transition pore in the heart

Significance The massive cell death, associated with a heart attack, is mainly due to disruption of mitochondrial membrane integrity upon activation of the mitochondrial permeability transition pore. Thus, it is important to understand how this pore is regulated to prevent cardiac cell death. In this study, we reported that hematopoietic-substrate-1 associated protein X-1 (HAX-1) is an inhibitor of the pore and promotes cell survival. HAX-1 works through recruitment of a chaperone protein called Hsp90 from cyclophilin-D, a major component of the pore. Displacement of Hsp90 from cyclophilin-D promotes cyclophilin-D degradation, resulting in inhibition of pore opening and cell death. Given that the opening of the mitochondrial permeability transition pore contributes to various diseases, our findings have broader applications reaching beyond the heart. The major underpinning of massive cell death associated with myocardial infarction involves opening of the mitochondrial permeability transition pore (mPTP), resulting in disruption of mitochondria membrane integrity and programmed necrosis. Studies in human lymphocytes suggested that the hematopoietic-substrate-1 associated protein X-1 (HAX-1) is linked to regulation of mitochondrial membrane function, but its role in controlling mPTP activity remains obscure. Herein we used models with altered HAX-1 expression levels in the heart and uncovered an unexpected role of HAX-1 in regulation of mPTP and cardiomyocyte survival. Cardiac-specific HAX-1 overexpression was associated with resistance against loss of mitochondrial membrane potential, induced by oxidative stress, whereas HAX-1 heterozygous deficiency exacerbated vulnerability. The protective effects of HAX-1 were attributed to specific down-regulation of cyclophilin-D levels leading to reduction in mPTP activation. Accordingly, cyclophilin-D and mPTP were increased in heterozygous hearts, but genetic ablation of cyclophilin-D in these hearts significantly alleviated their susceptibility to ischemia/reperfusion injury. Mechanistically, alterations in cyclophilin-D levels by HAX-1 were contributed by the ubiquitin-proteosomal degradation pathway. HAX-1 overexpression enhanced cyclophilin-D ubiquitination, whereas proteosomal inhibition restored cyclophilin-D levels. The regulatory effects of HAX-1 were mediated through interference of cyclophilin-D binding to heat shock protein-90 (Hsp90) in mitochondria, rendering it susceptible to degradation. Accordingly, enhanced Hsp90 expression in HAX-1 overexpressing cardiomyocytes increased cyclophilin-D levels, as well as mPTP activation upon oxidative stress. Taken together, our findings reveal the role of HAX-1 in regulating cyclophilin-D levels via an Hsp90-dependent mechanism, resulting in protection against activation of mPTP and subsequent cell death responses.

Up‐regulation of micro‐RNA765 in human failing hearts is associated with post‐transcriptional regulation of protein phosphatase inhibitor‐1 and depressed contractility

Impaired sarcoplasmic reticulum (SR) Ca2+ cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP‐1) activity, associated with down‐regulation of its endogenous inhibitor‐1. The levels and activity of inhibitor‐1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor‐1 by post‐transcriptional modification.

CXCR4 attenuates cardiomyocytes mitochondrial dysfunction to resist ischaemia-reperfusion injury

The chemokine (C‐X‐C motif) receptor 4 (CXCR4) is expressed on native cardiomyocytes and can modulate isolated cardiomyocyte contractility. This study examines the role of CXCR4 in cardiomyocyte response to ischaemia‐reperfusion (I/R) injury. Isolated adult rat ventricular cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. In response to H/R injury, the decrease in CXCR4 expression was associated with dysfunctional energy metabolism indicated by an increased adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio. CXCR4‐overexpressing cardiomyocytes were used to determine whether such overexpression (OE) can prevent bio‐energetic disruption‐associated cell death. CXCR4 OE was performed with adenoviral infection with CXCR4 encoding‐gene or non‐translated nucleotide sequence (Control). The increased CXCR4 expression was observed in cardiomyocytes post CXCR4‐adenovirus transduction and this OE significantly reduced the cardiomyocyte contractility under basal conditions. Although the same extent of H/R‐provoked cytosolic calcium overload was measured, the hydrogen peroxide‐induced decay of mitochondrial membrane potential was suppressed in CXCR4 OE group compared with control group, and the mitochondrial swelling was significantly attenuated in CXCR4 group, implicating that CXCR4 OE prevents permeability transition pore opening exposure to overload calcium. Interestingly, this CXCR4‐induced mitochondrial protective effect is associated with the enhanced signal transducer and activator of transcription 3 (expression in mitochondria. Consequently, in the presence of H/R, mitochondrial dysfunction was mitigated and cardiomyocyte death was decreased to 65% in the CXCR4 OE group as compared with the control group. I/R injury leads to the reduction in CXCR4 in cardiomyocytes associated with the dysfunctional energy metabolism, and CXCR4 OE can alleviate mitochondrial dysfunction to improve cardiomyocyte survival.

Repair Injured Heart by Regulating Cardiac Regenerative Signals

Cardiac regeneration is a homeostatic cardiogenic process by which the sections of malfunctioning adult cardiovascular tissues are repaired and renewed employing a combination of both cardiomyogenesis and angiogenesis. Unfortunately, while high-quality regeneration can be performed in amphibians and zebrafish hearts, mammalian hearts do not respond in kind. Indeed, a long-term loss of proliferative capacity in mammalian adult cardiomyocytes in combination with dysregulated induction of tissue fibrosis impairs mammalian endogenous heart regenerative capacity, leading to deleterious cardiac remodeling at the end stage of heart failure. Interestingly, several studies have demonstrated that cardiomyocyte proliferation capacity is retained in mammals very soon after birth, and cardiac regeneration potential is correspondingly preserved in some preadolescent vertebrates after myocardial infarction. There is therefore great interest in uncovering the molecular mechanisms that may allow heart regeneration during adult stages. This review will summarize recent findings on cardiac regenerative regulatory mechanisms, especially with respect to extracellular signals and intracellular pathways that may provide novel therapeutics for heart diseases. Particularly, both in vitro and in vivo experimental evidences will be presented to highlight the functional role of these signaling cascades in regulating cardiomyocyte proliferation, cardiomyocyte growth, and maturation, with special emphasis on their responses to heart tissue injury.

Manipulating the Hippo-Yap signal cascade in stem cells for heart regeneration

The Hippo-Yap pathway was originally recognized as a crucial signal cascade controlling organ size, and more recently identified as an important component involved in the regulation of cardiomyocyte survival, proliferation, and regeneration. Negative stress responses can activate mammalian sterile 20-like kinase 1 (Mst1) to suppress protective autophagy and promote cardiomyocyte apoptosis via phosphorylation and inhibition of Bcl-xL. Moreover, decreased Yap activity and nuclear entry will decrease upon Mst1 activation, ultimately suppressing cardiomyocytes proliferation and regeneration. Based on these observations, there are potential therapeutic opportunities in cardiac structural and functional regeneration post myocardium infarction to be gained by manipulation of the Hippo-Yap signal cascade. This review will summarize the main components of the Hippo-Yap pathway and their molecular biological functions. It will then highlight the role of these signal modules in the acquisition of stem cell pluripotency, cardiogenic differentiation, cardiomyocyte proliferation and maturation, and mitochondrial biogenesis in cardiac stem cells. Finally, it will discuss the potential for future studies of Hippo-Yap pathway using induced pluripotent stem cell (iPSC) technology.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia

Significance A common variant of histidine-rich Ca-binding protein (HRC), where an alanine replaces a serine at amino acid 96, can increase the risk of dying from severe heart disease. Using human, mice, and cellular models, we show that this variant blocks position 96 from becoming phosphorylated, a prevalent type of protein modification carried out by kinase enzymes. We demonstrate that phosphorylation of HRC at Ser96 indeed provides protection from heart disease, and we identify family with sequence similarity 20C (Fam20C) as the kinase that phosphorylates HRC. HRC phosphorylation appears to play a role in regulating Ca cycling that is critical for proper cardiac muscle contraction. This demonstration of Fam20C’s role in heart disease opens up avenues for potential preventative or therapeutic strategies. Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6S10F mutant

ABSTRACT 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 (HSPB6S10F) 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 HSPB6S10F 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 HSPB6S10F-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.

A novel human S10F‐Hsp20 mutation induces lethal peripartum cardiomyopathy

Heat shock protein 20 (Hsp20) has been shown to be a critical regulator of cardiomyocyte survival upon cardiac stress. In this study, we investigated the functional significance of a novel human Hsp20 mutation (S10F) in peripartum cardiomyopathy. Previous findings showed that cardiac‐specific overexpression of this mutant were associated with reduced autophagy, left ventricular dysfunction and early death in male mice. However, this study indicates that females have normal function with no alterations in autophagy but died within a week after 1‐4 pregnancies. Further examination of mutant females revealed left ventricular chamber dilation and hypertrophic remodelling. Echocardiography demonstrated increases in left ventricular end‐systolic volume and left ventricular end‐diastolic volume, while ejection fraction and fractional shortening were depressed following pregnancy. Subsequent studies revealed that cardiomyocyte apoptosis was elevated in mutant female hearts after the third delivery, associated with decreases in the levels of Bcl‐2/Bax and Akt phosphorylation. These results indicate that the human S10F mutant is associated with dysregulation of cell survival signalling, accelerated heart failure and early death post‐partum.