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Background LIVE (Less Invasive Ventricular Enhancement) with Revivent TC™ is an innovative therapy for symptomatic ischemic Heart Failure (HF). It is designed to reconstruct a negatively remodeled left ventricle (LV) after an anterior myocardial infarction (MI) by plication of the scar tissue. Its indications are specific and, as with any other structural heart intervention, the success of the procedure starts with appropriate patient selection. We aim to present the indications of the technique, crucial aspects in patient selection and individual case planning approach. Methods and Results After clinical evaluation, transthoracic echocardiography is the first imaging modality to be performed in a potential candidate for the therapy. However, definitive indication and detailed case planning rely on late gadolinium-enhanced cardiac magnetic resonance imaging or multiphasic contrast-enhanced cardiac computed tomography. These imaging modalities also assist with relative or absolute contra-indications for the procedure. Individual assessment is done to tailor the procedure to the specifics of the LV anatomy and location of the myocardial scar. Conclusion LIVE procedure is a unique intervention to treat symptomatic heart failure and ischemic cardiomyopathy after anterior MI. It is a highly customizable intervention that allows a patient-tailored approach, based on multimodality imaging assessment and planification.

Key message We mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome primarily responsible for inhibiting gravitropism, proving the utility of the mutant library. Abstract The CRISPR-Cas9 system is a powerful tool for genome editing. Here we report highly-efficient multiplex CRISPR-Cas9 editing of the seven-member phytochrome gene family in the model bryophyte Physcomitrium (Physcomitrella) patens. Based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to identify high-order multiple mutants prior to sequencing, we demonstrate successful targeting of all seven PHY genes in a single transfection. We investigated further aspects of the CRISPR methodology in Physcomitrella, including the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repairing the chromosome when excising a complete locus. As proof-of-principle, we show that the septuple phy− mutant remains gravitropic in light, in line with expectations, and on the basis of data from lower order multiplex knockouts conclude that phy5a is the principal phytochrome responsible for inhibiting gravitropism in light. We expect, therefore, that this mutant collection will be valuable for further studies of phytochrome function and that the methods we describe will allow similar approaches to revealing specific functions in other gene families.

Objectives: Ischemic cardiomyopathy is the principal cause of heart failure. In patients with left ventricular (LV) dilatation,low ejection fraction (EF), and transmural scar in anteroseptal distribution, surgical ventricular reconstruction (SVR) can beconsidered, although it is an invasive surgical procedure. Less Invasive Ventricular Enhancement (LIVE) technique emergedas a unique intervention to exclude scarred myocardium, improving symptoms and quality of life. We aim to present LIVEcontemporary short and mid-term outcomes. Methods: LIVE procedure has evolved from open sternotomy to a hybrid procedure done with right internal jugular veinaccess and a left minithoracotomy. LV shape and size are restored without extracorporeal circulation by plication of thescarred myocardium. This is achieved by implantation of a series of internal and external microanchors brought togetherover a poly-ether-ether-ketone (PEEK) tether to make a longitudinal approximation between the LV free wall and theanterior septum. Internal anchors are deployed by a transcatheter technique, through the right internal jugular vein, on theright side of the ventricular septum. Result: Between July 2018 and August 2020, a total of 71 patients (84.5% men;mean age 61 ± 12.3 years) weresubmitted to the LIVE procedure in 18 institutions in Europe, North America, and Asia. Procedural success was 100%. Amean of 2.4 anchor pairs (median 3) was used to reshape the LV. Echocardiographic data showed an increase in LV EF from31.0 ± 9.2 to 38.9 ± 12.6% (change +29.8%, p < 0.001) and LV end-systolic volume index (LVESVI) reduction from 68.0 ± 28.8 mL/m2 to 42.3 ± 20.7 mL/m2 (change: -37.9%, p < 0.001) after the procedure. No sternotomy conversion was needed.New onset tricuspid valve regurgitation was observed in one patient. There was no case of ventricular septal defect.Observed mortality was 2.8% (2 patients): one due to severe contrast dye induced anaphylactic shock and another one dueto COVID-19. In the follow-up, NYHA class improved a median of 1 grade and there was no late mortality. Conclusion: Hybrid LV reshaping and volume reduction has proven to be a useful solution for patients with symptomaticheart failure after left anterior descending territory myocardial infarction. These results from the latest iteration of thetechnique show that this approach is safe, reproducible, and has a significant short and mid-term impact on improving EFand reducing LV size.

In mosses such as Physcomitrella patens phytochrome photoreceptors steer directional/vectorial responses to unilateral/polarized light. In this chapter, we describe procedures to assay phototropism and polarotropism quantitatively in wild type and mutant lines. Protonemata are placed on agar-based medium in square Petri dishes in darkness for 1 week, allowing caulonemata to develop and grow negatively gravitropically. For phototropism, the dishes are placed vertically in black boxes and unilaterally irradiated with continuous red light. For polarotropism, Petri dishes are placed horizontally and irradiated with linearly polarized red light from above. After irradiation, the filaments are photographed using a macroscope with CCD camera and the bending angles measured using image processing software. The data are transfered to a spreadsheet program, placed into 10° bending angle classes and illustrated using a circular histogram.

Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.

International audience; Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.

In mosses such as Physcomitrella patens phytochrome photoreceptors steer directional/vectorial responses to unilateral/polarized light. In this chapter, we describe procedures to assay phototropism and polarotropism quantitatively in wild type and mutant lines. Protonemata are placed on agar-based medium in square Petri dishes in darkness for 1 week, allowing caulonemata to develop and grow negatively gravitropically. For phototropism, the dishes are placed vertically in black boxes and unilaterally irradiated with continuous red light. For polarotropism, Petri dishes are placed horizontally and irradiated with linearly polarized red light from above. After irradiation, the filaments are photographed using a macroscope with CCD camera and the bending angles measured using image processing software. The data are transfered to a spreadsheet program, placed into 10° bending angle classes and illustrated using a circular histogram.