AAV9.I-1c Delivered via Direct Coronary Infusion in a Porcine Model of Heart Failure Improves Contractility and Mitigates Adverse Remodeling

Heart failure is characterised by impaired function and disturbed Ca2+ homeostasis. Transgenic increases in inhibitor-1 activity have been shown to improve Ca2 cycling and preserve cardiac performance in the failing heart. The aim of this study is to evaluate the effect of activating the inhibitor (I-1c) of protein phosphatase 1 (I-1) through gene transfer on cardiac function in a porcine model of heart failure induced by myocardial infarction (MI)

EphA2 Interacts with Tim-4 through Association between Its FN3 Domain and the IgV Domain of Tim-4

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa

The TSP-1 domain of the matricellular protein CCN5 is essential for its nuclear localization and anti-fibrotic function.

The matricellular protein CCN5 exerts anti-fibrotic activity in hearts partly by inducing reverse trans-differentiation of myofibroblasts (MyoFBs) to fibroblasts (FBs). CCN5 consists of three structural domains: an insulin-like growth factor binding protein (IGFBP), a von Willebrand factor type C (VWC), and a thrombospondin type 1 (TSP-1). In this study, we set out to elucidate the roles of these domains in the context of the reverse trans-differentiation of MyoFBs to FBs. First, human cardiac FBs were trans-differentiated to MyoFBs by treatment with TGF-β; this was then reversed by treatment with recombinant human CCN5 protein or various recombinant proteins comprising individual or paired CCN5 domains. Subcellular localization of these recombinant proteins was analyzed by immunocytochemistry, cellular fractionation, and western blotting. Anti-fibrotic activity was also evaluated by examining expression of MyoFB-specific markers, α-SMA and fibronectin. Our data show that CCN5 is taken up by FBs and MyoFBs mainly via clathrin-mediated endocytosis, which is essential for the function of CCN5 during the reverse trans-differentiation of MyoFBs. Furthermore, we showed that the TSP-1 domain is essential and sufficient for endocytosis and nuclear localization of CCN5. However, the TSP-1 domain alone is not sufficient for the anti-fibrotic function of CCN5; either the IGFBP or VWC domain is needed in addition to the TSP-1 domain

Effects of mechanical properties of gelatin methacryloyl hydrogels on encapsulated stem cell spheroids for 3D tissue engineering

Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (<1 kPa), has not yet been studied. In this study, we determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction

AAV-Mediated Knock-Down of HRC Exacerbates Transverse Aorta Constriction-Induced Heart Failure

<div><h3>Background</h3><p>Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca²⁺ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca²⁺ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca²⁺ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.</p> <h3>Methodology/Principal Findings</h3><p>AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca²⁺ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca²⁺ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.</p> <h3>Conclusions/Significance</h3><p>Increased Ca²⁺ leak and cytosolic Ca²⁺ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca²⁺ cycling.</p> </div

siRNA-mediated HRC knock-down (KD) and the expressional profiles of SR proteins in the control and KD samples.

<p>siNC and siHRC oligonucleotides (Dharmacon) were transfected to neonatal rat ventricular cells (NRVCs). After 48 h, transfected NRVCs were solubilized with 1% SDS lysis buffer, and western blot analyses were performed using various antibodies. A: Western blot result of SR proteins in NRVCs after HRC-KD. RyR, ryanodine receptor; HRC, histidine-rich calcium binding protein; SERCA2a, sarcoplasmic reticulum Ca²⁺ ATPase 2a; DHPR, dihydropyridine receptor; NCX, Na⁺-Ca²⁺ exchanger; CSQ, calsequestrin; CaMKII, Ca²⁺/calmodulin-dependent kinase; TRN, triadin. B: Relative expression levels of SR proteins after HRC-KD. siNC, negative control of knock-down oligonucleotide; siHRC, HRC-KD oligonucleotide (**<i>P</i><0.01). Note that there were no expressional changes of other SR proteins by knock-down of HRC.</p

AAV9 virus -mediated HRC-KD in sham and TAC-FH samples.

<p>A: Schematic figure for the construction of shNC- and shHRC-AAV9 viruses. B: Heart weight per body weight (HW/BW) ratio after 11 weeks of TAC operation. C: Heart weight per tibia length (HW/TL) ratio after 11 weeks of TAC operation. D: Western blot result of HRC expression from 4 groups, shNC sham, shHRC sham, shNC failure and shHRC failure. E: Summarized data for HRC expression (*<i>P</i><0.05, **<i>P</i><0.01). Note that the HRC expression was down-regulated both in sham and failure samples.</p

Severely down-regulated cardiac function and cardiac fibrosis in HRC-KD heart under heart failure condition.

<p>A: Representative M-mode echocardiograms of all groups. Echocardiography on the experimental animals was performed at 1, 5, 9, and 11 weeks after sham or TAC operation. B: Changes in fractional shortening after viral injection for the duration of 11 weeks in sham- and TAC-operated shNC and shHRC mice. HRC-KD heart showed most severely down-regulated fractional shortening (FS) under heart failure condition. C: Masson’s trichrome staining results. Blue color indicates the appearance of collagen; red color indicates that the cytoplasmic area is occupied by myocytes and erythrocytes; black color indicates the presence of nuclei. HRC-KD heart showed most severe fibrosis under heart failure condition. shNC, negative control of AAV9; shHRC, HRC-KD through AAV9 (*<i>P</i><0.05).</p