Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction.

Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from ß-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under ß-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ß-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

RATIONALE: Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. OBJECTIVE: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. METHODS AND RESULTS: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. CONCLUSIONS: Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.