o-ring-aortic-banding-versus-traditional-transverse-aortic

O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of ...

University Hospital Kiel, Partner Site Hamburg/Kiel/Lübeck

Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective.

Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in cardiac hypertrophy and failure.

Uromodulin is produced exclusively in the kidney and secreted into both urine and blood. Serum levels of uromodulin are correlated with kidney function and reduced in chronic kidney disease (CKD) patients, but physiological functions of serum uromodulin are still elusive. This study investigated the role of uromodulin in medial vascular calcification, a key factor associated with cardiovascular events and mortality in CKD patients.

Circulating uromodulin inhibits vascular calcification by interfering with pro-inflammatory cytokine signalling.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.

Gene therapeutic approaches to aortic diseases require efficient vectors and delivery systems for transduction of endothelial cells (ECs) and smooth muscle cells (SMCs). Here, we developed a novel strategy to efficiently deliver a previously described vascular-specific adeno-associated viral (AAV) vector to the abdominal aorta by application of alginate hydrogels. To efficiently transduce ECs and SMCs, we used AAV9 vectors with a modified capsid (AAV9SLR) encoding enhanced green fluorescent protein (EGFP), as wild-type AAV vectors do not transduce ECs and SMCs well. AAV9SLR vectors were embedded into a solution containing sodium alginate and polymerized into hydrogels. Gels were surgically implanted around the adventitia of the infrarenal abdominal aorta of adult mice. Three weeks after surgery, an almost complete transduction of both the endothelium and tunica media adjacent to the gel was demonstrated in tissue sections. Hydrogel-mediated delivery resulted in induction of neutralizing antibodies but did not cause inflammatory responses in serum or the aortic wall. To further determine the translational potential, aortic tissue from patients was embedded  into AAV9SLR-containing hydrogel, and efficient transduction could be confirmed. These findings demonstrate that alginate hydrogel harboring a vascular-targeting AAV9SLR vector allows efficient local transduction of the aortic wall.

Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue.

Autophagy is a cellular degradation process that has been implicated in diverse disease processes. The authors provide evidence that FYCO1, a component of the autophagic machinery, is essential for adaptation to cardiac stress. Although the absence of FYCO1 does not affect basal autophagy in isolated cardiomyocytes, it abolishes induction of autophagy after glucose deprivation. Likewise, -deficient mice subjected to starvation or pressure overload are unable to respond with induction of autophagy and develop impaired cardiac function. FYCO1 overexpression leads to induction of autophagy in isolated cardiomyocytes and transgenic mouse hearts, thereby rescuing cardiac dysfunction in response to biomechanical stress.

FYCO1 Regulates Cardiomyocyte Autophagy and Prevents Heart Failure Due to Pressure Overload In Vivo.

Fibroblast growth factor (FGF) 23 is elevated in chronic kidney disease (CKD) to maintain phosphate homeostasis. FGF23 is associated with left ventricular hypertrophy (LVH) in CKD and induces LVH  klotho-independent FGFR4-mediated activation of calcineurin/nuclear factor of activated T cells (NFAT) signaling in animal models, displaying systemic alterations possibly contributing to heart injury. Whether elevated FGF23  causes LVH in healthy animals is unknown. By generating a mouse model with high intra-cardiac Fgf23 synthesis using an adeno-associated virus (AAV) expressing murine Fgf23 (AAV-Fgf23) under the control of the cardiac troponin T promoter, we investigated how cardiac Fgf23 affects cardiac remodeling and function in C57BL/6 wild-type mice. We report that AAV-Fgf23 mice showed increased cardiac-specific  mRNA expression and synthesis of full-length intact Fgf23 (iFgf23) protein. Circulating total and iFgf23 levels were significantly elevated in AAV-Fgf23 mice compared to controls with no difference in bone  expression, suggesting a cardiac origin. Serum of AAV-Fgf23 mice stimulated hypertrophic growth of neonatal rat ventricular myocytes (NRVM) and induced pro-hypertrophic NFAT target genes in klotho-free culture conditions . Further analysis revealed that renal Fgfr1/klotho/extracellular signal-regulated kinases 1/2 signaling was activated in AAV-Fgf23 mice, resulting in downregulation of sodium-phosphate cotransporter  and  and suppression of , further supporting the bioactivity of cardiac-derived iFgf23. Of interest, no LVH, LV fibrosis, or impaired cardiac function was observed in klotho sufficient AAV-Fgf23 mice. Verified in NRVM, we show that co-stimulation with soluble klotho prevented Fgf23-induced cellular hypertrophy, supporting the hypothesis that high cardiac Fgf23 does not act cardiotoxic in the presence of its physiological cofactor klotho. In conclusion, chronic exposure to elevated cardiac iFgf23 does not induce LVH in healthy mice, suggesting that Fgf23 excess  does not tackle the heart.

Cardiac Fibroblast Growth Factor 23 Excess Does Not Induce Left Ventricular Hypertrophy in Healthy Mice.

The sarcoplasmic reticulum (SR) Ca-ATPase 2 (SERCA2) mediates Ca reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca release from SR and triggers contraction. Ca/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.

Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Article	Year
Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in cardiac hypertrophy and failure. Cardiovascular research\| PubMed ID: 30418544	2019
Circulating uromodulin inhibits vascular calcification by interfering with pro-inflammatory cytokine signalling. Cardiovascular research\| PubMed ID: 32243494	2021
Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nature communications\| PubMed ID: 33116134	2020
Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue. Molecular therapy. Methods & clinical development\| PubMed ID: 33768132	2021
FYCO1 Regulates Cardiomyocyte Autophagy and Prevents Heart Failure Due to Pressure Overload In Vivo. JACC. Basic to translational science\| PubMed ID: 33997522	2021
Cardiac Fibroblast Growth Factor 23 Excess Does Not Induce Left Ventricular Hypertrophy in Healthy Mice. Frontiers in cell and developmental biology\| PubMed ID: 34778257	2021
AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR. Circulation research\| PubMed ID: 34814703	2022
Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs. Basic research in cardiology\| PubMed ID: 35737129	2022
Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. Science advances\| PubMed ID: 36129972	2022

Susanne Hille

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