| 1. |
- De Genst, Erwin, et al.
(författare)
-
Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure
- 2022
-
Ingår i: Nature Communications. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 2041-1723.
-
Tidskriftsartikel (refereegranskat)abstract
- The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
|
|
| 2. |
- Rohner, Eduarde
(författare)
-
A modular mRNA approach to study and treat phospholamban mediated cardiomyopathies
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the heart, phospholamban (PLN) is a key regulator of calcium cycling through its inhibitory effect on the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. For more than two decades, modulating calcium channels has been a strong focus of prospective cardiac therapeutics with a recent emphasis on targeting PLN itself, partially due to the progressive uncovering of several cardiomyopathic PLN human mutations. Our understanding of the physiological and pathophysiological mechanisms regulating or misregulating calcium cycling in humans in not fully understood. Therefore, careful study of naturally occurring human mutations can offer important insights into these mechanisms. This thesis focuses on developing novel investigative and potential therapeutic tools to mechanically dissect and treat phospholamban mediated cardiomyopathies. The constituent papers compiled in this thesis validates these novel and innovative approaches which utilize an mRNA modular system to model and dissect core mechanistic differences caused by cardiomyopathic phospholamban mutations in embryonic stem cell derived cardiomyocytes. Additionally, this thesis showcases the usage of single-chain VHH antibodies (intrabodies) to express, target and modulate PLN intracellularly in vitro and in vivo with promising results validating its usage as a potential therapy and valuable investigative tool.
|
|
| 3. |
- Rohner, Eduarde, et al.
(författare)
-
An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation
- 2021
-
Ingår i: Molecular Medicine. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1528-3658 .- 1076-1551.
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. Methods: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. Results: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. Conclusion: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.
|
|
