Notes on the last day of the conference. For me the highlight was the Dylan Burnette’s talk on sarcomere assembly, something I can directly relate to. Too much good stuff.

Giulia Savore

The Melusin cardioprotective chaperone tunes lipid metabolism to promote cardiac resilience to stress
University of Torino, Italy

Melusin one interacts with Integrin-beta one and also with mitochondria. It stimulates compensatory hypertrophy and protects from oxidative stress.

Dylan T. Burnette

Regulation of ventricular contractility driven atrial ballooning by the alpha actinin family member ACTN4
Vanderbilt University, United States of America

Models of sarcomere assembly revisited

The talk started with a history of sarcomere assembly, there are two main schools of thought. FIrst the template model, then the Pre-myofibril model which is the one they think is relevant. Basically it means that the myofibril assembles form non-muscle molecules (like non muscle myosin II B), get some organization and later they are replaced by the muscle specific proteins.

ACTN4

SO they decided to overexpress ACTN4-GFP to see whether it is also localized in sarcomeric structures and it is. Using alpha fold they predicted that it should interact with the proteins in the Z-disc. Indeed they show that when they silence Actinin4 the sarcomere formation is improved. The cells get bigger, Z-disc number increases and myofibrils are thicker. Not sure what happens when they overexpress it. Is probably in the paper.

Myosin activator and inhibitor

Papers

Eva van Rooij

New concepts to treat arrhythmogenic cardiomyopathy
Hubrecht Institute, The Netherlands

Interested in arrythmogenic cardiomyopathy (cause of sudden cardiac death) with a frequency of 1:1000-5000, which is called the disease of a desmosome, as the defective connection between adjacent cardiomyocytes is to blame for the arrythmias. Plakophilin (PKP2) is mutated in many patients.\

Did correlation of PKP2 levels with other components of desmosome and found that they are correlecting; PKP2 is essential for a good functional desmosome. The other genes were JUP, DSP, DSG, DSC. This phenotype can be rescued using aav6 overexpressed PKP2.

THe presentation took us from hiPSCs through mouse to big animal model including several companies on the way. To cut the story short, they were able to get to 80% efficiency of PKP2 reexpression.

Q/A

  • ONe of the very interesting notions was that there is some internal level of control of gene expression, you cannot push PKP2 higher then normal. THe overexpression dilutes out the mutant variant, but thats it. Was mentioned from the audience, that this is typical for structural proteins in cardiomyocytes.
  • aavs are not the best for therapy, 20-30% of people have antibodies, companies working on this actively

Mehdiabadi

Re-evaluating the fetal gene program at single-cell resolution in dilated cardiomyopathy (DCM)
Murdoch Children’s Research Institute, Australia

What is the foetal gene expression programme?

The concept of reactivation of foetal gene reexpression in hypertrophy and disease is already 40 years plus old. They decided to have a fresh look at the problem using five groups of samples.

  1. foetal hearts
  2. healthy hearts from kids
  3. dcm hearts from kids
  4. healthy hearts from adults
  5. dcm hearts from adults

They did an intersection of genes expressed in foetal hearts and intersection of pediatric healthy and control.

This gives 163 genes which can be considered the reactivated fetal genes. Then they compared these genes in pediatric and adult dcm.

She did not really show much more but mentioned that the genes in the datasets are mostly shared between fibroblasts and cardiomyocytes, cell adhesion goes up, immune response goes down. Very interesting, but at the end, very little information to use.

Papers

Mehdiabadi..Porello 2022, Circulation

Lars Steinmetz

Regulation of splicing in cardiomyopathies
Stanford University School of Medicine, United States/EMBL Heidelberg, Germany

RBM20 as the key player

DIlated cardiomyopathy is associated with RBM20 splicing protein mutations. In particular there is a hotspot of mutations in RS domain. The most famous mutation in RBM20 RS domain is P633L(?) which is associated with a wrong splicing of CAMKIID, RYR2, TTN, and so on.

HIPSCs derived cardiomyocytes with this mutation have icnreased calcium intake, impaired slicing, and increased contractility (which kills them, simialr to duschenne which are more contractile, than controls).

They used Nanopore for sequencing of cDNA to see what is the distribution of the isofomrs (Zhu, 2021).

RBM20 can be localized in three principal places

They did a huge screen to see the effects of the localization of RBM20 (basically changed all AAs in the hotspot (16? AAs) and highthroughput to see the localization of RBM20 (image assisted cell-sorting). INterestlingly the 633 mutation was more severe than KO. Also identified the major transporter of RBM20 (TNPO3)

Works in vitro

Eventually they used the knowledge about what aminoacid changes make what effect in vivo in mouse using the myoaavs and worked good.

Q/A

  • no localization of RBM20 on sarcomeres, diffuse in sarcoplasm when inactive, ohtherwise in nucleus speckles okey
  • also see the perinculear localization but not included here

Papers

Eric Olson

Toward the genetic correction of heart and muscle disease
UT Southwestern Medical Center, United States of America

They use different platforms of muscle: skeletal, cardiac and smooth.

Duchenne intro

Dystrophin gene is super long (takes 16hours to transcribe to mRNA). There is a lot of exons and some of them need to fit into another (like a puzzle) while some have blunt edges so any can fit together well. Therefore when one of the “pouzzle exons are damaged, the protein does not get produced at all. But if we cut out the neighbouring two puzzle pieces, the rest will fit by blunt edges and the protein will be produced. This is called “exon skipping”.

Three types of CRISPR to fix mutations

  1. Clasical crispr uses gRNA to cut sequence and then use NHEJ to glue it together
  2. Single base editing - enables change just one base, so this is much more specific
  3. Multiple base editing - don’t know how but changes multiple bases in one go

Application-01 Duchenne

  • use exon skipping when exon 44 is faulty, cut out 45 and it works
  • also did it in dogs (there is some mutated breed in london veterinary hospital) and it works

For DCM

  • used on RBM20 correction
  • used for TTN correction
  • used for Myh7

Application for ischemic

Well after being able to fix inherited diseases, what about fix lifestyle diseasee? CAMKII-delta phosphorylated HAC4 and leads to dilated cardiomyopathy. So they mutated the residues on CAMKII-delta responsible for HDAC4 phosphorylation and show that it then fails to activate HDAC4 and thus reverts the disease. Kept the mouse for whole life, challenged by excercise etc. but the mice were okey.

This is supr cool

Q/A

  • How many cells need to be corrected for rescue? 60% is okey
  • Can endogenous rna modifying enzymes be leveraged in future to do the same? ???

Papers