Hyaloid Vasculature and mmp2 Activity Play a Role during Optic Fissure Fusion in Zebrafish

Vertebrate retinal development requires timely and precise fusion of the optic fissure (OF). Failure of this event leads to congenital vision impairment in the form of coloboma. Recent studies have suggested hyaloid vasculature to be involved in OF fusion. In order to examine this link, we analyzed OF fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a−/− embryos fail to accumulate F-actin in the OF prior to basement membrane (BM) degradation. Furthermore, using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a−/− embryos. When examining the connection between pax2a loss of function and hyaloid vasculature, we observed significant reduction of talin1 expression, a regulator of hyaloid vasculature. In addition, cranial VEGF expression was found to be reduced in pax2a−/− embryos. Pharmacological inhibition of VEGF signaling phenocopied the pax2a−/− vasculature, F-actin and BM degradation phenotypes. Lastly, we determined that OF associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of OF BM. Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF in order to facilitate availability of the BM remodeler mmp2

Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS

Aerobic Exercise Training Prevents Heart Failure-Induced Skeletal Muscle Atrophy by Anti-Catabolic, but Not Anabolic Actions

Background: Heart failure (HF) is associated with cachexia and consequent exercise intolerance. Given the beneficial effects of aerobic exercise training (ET) in HF, the aim of this study was to determine if the ET performed during the transition from cardiac dysfunction to HF would alter the expression of anabolic and catabolic factors, thus preventing skeletal muscle wasting. Methods and Results: We employed ascending aortic stenosis (AS) inducing HF in Wistar male rats. Controls were sham operated animals. At 18 weeks after surgery, rats with cardiac dysfunction were randomized to 10 weeks of aerobic ET (AS-ET) or to an untrained group (AS-UN). At 28 weeks, the AS-UN group presented HF signs in conjunction with high TNF-α serum levels; soleus and plantaris muscle atrophy; and an increase in the expression of TNF-α, NFkB (p65), MAFbx, MuRF1, FoxO1, and myostatin catabolic factors. However, in the AS-ET group, the deterioration of cardiac function was prevented, as well as muscle wasting, and the atrophy promoters were decreased. Interestingly, changes in anabolic factor expression (IGF-I, AKT, and mTOR) were not observed. Nevertheless, in the plantaris muscle, ET maintained high PGC1α levels. Conclusions: Thus, the ET capability to attenuate cardiac function during the transition from cardiac dysfunction to HF was accompanied by a prevention of skeletal muscle atrophy that did not occur via an increase in anabolic factors, but through anti-catabolic activity, presumably caused by PGC1α action. These findings indicate the therapeutic potential of aerobic ET to block HF-induced muscle atrophy by counteracting the increased catabolic state

Aerobic Exercise Training Prevents Heart Failure-Induced Skeletal Muscle Atrophy by Anti-Catabolic, but Not Anabolic Actions

Background: Heart failure (HF) is associated with cachexia and consequent exercise intolerance. Given the beneficial effects of aerobic exercise training (ET) in HF, the aim of this study was to determine if the ET performed during the transition from cardiac dysfunction to HF would alter the expression of anabolic and catabolic factors, thus preventing skeletal muscle wasting.Methods and Results: We employed ascending aortic stenosis (AS) inducing HF in Wistar male rats. Controls were sham-operated animals. At 18 weeks after surgery, rats with cardiac dysfunction were randomized to 10 weeks of aerobic ET (ASET) or to an untrained group (AS-UN). At 28 weeks, the AS-UN group presented HF signs in conjunction with high TNF-alpha serum levels; soleus and plantaris muscle atrophy; and an increase in the expression of TNF-alpha, NF kappa B (p65), MAFbx, MuRF1, FoxO1, and myostatin catabolic factors. However, in the AS-ET group, the deterioration of cardiac function was prevented, as well as muscle wasting, and the atrophy promoters were decreased. Interestingly, changes in anabolic factor expression (IGF-I, AKT, and mTOR) were not observed. Nevertheless, in the plantaris muscle, ET maintained high PGC1 alpha levels.Conclusions: Thus, the ET capability to attenuate cardiac function during the transition from cardiac dysfunction to HF was accompanied by a prevention of skeletal muscle atrophy that did not occur via an increase in anabolic factors, but through anti-catabolic activity, presumably caused by PGC1a action. These findings indicate the therapeutic potential of aerobic ET to block HF-induced muscle atrophy by counteracting the increased catabolic state.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Regulation of cardiac microRNAs induced by aerobic exercise training during heart failure

Exercise training (ET) has beneficial effects on the myocardium in heart failure (HF) patients and in animal models of induced cardiac hypertrophy and failure. We hypothesized that if microRNAs (miRNAs) respond to changes following cardiac stress, then myocardial profiling of these miRNAs may reveal cardio-protective mechanisms of aerobic ET in HF. We employed ascending aortic stenosis (AS) inducing HF in Wistar rats. Controls were sham-operated animals. At 18 weeks after surgery, rats with cardiac dysfunction were randomized to 10 weeks of aerobic ET (HF-ET) or to a heart failure sedentary group (HF-S). ET attenuated cardiac remodeling, as well as clinical and pathological signs of HF with maintenance of systolic and diastolic function when compared to HF-S. Global miRNA expression profiling of the cardiac tissue revealed 56 miRNAs differentially regulated in animals in the HF-ET, but only 12 miRNAs were differentially regulated in the HF-S. Out of 23 miRNAs that were differentially regulated in both groups, 17 miRNAs exhibited particularly high increases in expression, including miR-598, miR-429, miR-224, miR-425, and miR-221. From the initial set of deregulated miRNAs, 14 miRNAs with validated targets expressed in cardiac tissue that respond robustly to ET in HF were used to construct a miRNA-mRNA regulatory networks that revealed a set of 203 miRNA-target genes involved in programmed cell death, TGF-β signaling, cellular metabolic processes, cytokine signaling, and cell morphogenesis. Our findings reveal that ET attenuates cardiac abnormalities during HF by regulating cardiac miRNAs with potential role in cardio-protective mechanisms through multiple effects on gene expression.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e tecnológico (CNPq

Cross sectional area (µm²) of the two major soleus muscle fiber types after ET.

<p>Data are expressed as mean ± SD.</p><p>*p<0.05 vs. Sham-UN;</p>†<p>p<0.05 vs. AS-UN.</p><p>One-way ANOVA + Tukey test.</p><p>Cross sectional area (µm²) of the two major soleus muscle fiber types after ET.</p

Cross sectional area (µm²) of plantaris muscle fiber types after ET.

<p>Data are expressed as mean ± SD.</p><p>*p<0.05 vs. Sham-UN;</p>†<p>p<0.05 vs. AS-UN.</p><p>One-way ANOVA + Tukey test.</p><p>Cross sectional area (µm²) of plantaris muscle fiber types after ET.</p

Serial cross sections of the rat hindlimb muscles.

<p>Soleus (a) and plantaris (b, c and d) muscles taken from a Sham-UN group rat demonstrating fiber-type delineation as determined by the myofibrillar adenosine triphosphatase (mATPase) reaction after preincubation at pH 4.2 (b), 4.5 (a, c), and 10.6 (d). (I, type I; A, type IIA; D, type IID; and B, type IIB).</p

Serum concentration.

<p>Proinflammatory cytokine TNF-α (A) and growth factor IGF-I (B) in AS-UN, AS-ET, Sham-UN, and Sham-ET groups. Data are presented as the mean ± SD. *p<0.05 vs. Sham-UN.</p

Echocardiografic data before and after exercise training.

<p>HR, heart rate; LVDD and LVSD, LV diastolic and systolic diameters, respectively; PWT, LV posterior wall thickness; RWT, LV relative wall thickness in diastole; LA, left atrium diameter; AO, aortic diameter; FS, LV endocardial fractional shortening; PWSV, LV posterior wall shortening velocity; E/A, early-to-late diastolic mitral inflow ratio; EF, ejection fraction. n = 8 per group. Data are expressed as mean ± SD.</p><p>*p<0.05 vs. Sham-UN and,</p>#<p>p<0.05 vs. Sham-ET at the same time (18 wk or 28 wk);</p>†<p>p<0.05 vs. AS-UN;</p>‡<p>p<0.05 vs. AS; t test, one-way or 2-way ANOVA with Bonferroni <i>post hoc</i> test as appropriate.</p><p>Echocardiografic data before and after exercise training.</p