Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy
Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy
Florian Hinze 0 1 2 3
Christoph Dieterich 0 1 2 3
Michael H. Radke 0 1 2 3
Henk Granzier 0 1 2 3
Michael Gotthardt 0 1 2 3
0 DZHK (German Center for Cardiovascular Research) , partner site Berlin, Berlin , Germany
1 Neuromuscular and Cardiovascular Cell Biology, Max Delbrück Center for Molecular Medicine , Robert-Rössle-Str. 10, 13125 Berlin , Germany
2 DZHK (German Center for Cardiovascular Research) , partner site Heidelberg, Heidelberg , Germany
3 Klaus Tschira Institute for Integrative Computational Cardiology and Department of Cardiology, Angiology, and Pneumology, Heidelberg University , Analysezentrum III, INF 669, 69120 Heidelberg , Germany
Impaired diastolic filling is a main contributor to heart failure with preserved ejection fraction (HFpEF), a syndrome with increasing prevalence and no treatment. Both collagen and the giant sarcomeric protein titin determine diastolic function. Since titin's elastic properties can be adjusted physiologically, we evaluated titin-based stiffness as a therapeutic target. We adjusted RBM20-dependent cardiac isoform expression in the titin N2B knockout mouse with increased ventricular stiffness. A ~50 % reduction of RBM20 activity does not only maintain cardiac filling in diastole but also ameliorates cardiac atrophy and thus improves cardiac function in the N2Bdeficient heart. Reduced RBM20 activity partially normalized gene expression related to muscle development and fatty acid metabolism. The adaptation of cardiac growth was related to hypertrophy signaling via four-and-a-half lim-domain proteins (FHLs) that translate mechanical input into hypertrophy signals. We provide a novel link between cardiac isoform expression and trophic signaling via FHLs and suggest cardiac splicing as a therapeutic target in diastolic dysfunction.
Heart failure; Therapy; Mouse models; RNA processing; Hypertrophy signaling
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Cardiovascular disease is the main cause of death worldwide
with increasing prevalence of heart failure [1]. Multiple
environmental and genetic factors contribute to heart failure
including age, sex, diabetes, kidney disease, inflammation, and
mutations in sarcomeric proteins such as titin or cardiac splice
factors such as the RNA binding motif 20 (RBM20) that
regulates titin-based stiffness [2]. The giant sarcomeric protein
titin contributes to the diastolic properties of the heart. Titin
undergoes extensive posttranslational modifications and
alternative splicing adapts its elastic properties to the demands of
the organism [3, 4]. The elastic PEVK and N2B regions
support diastolic function, while differentially affecting cardiac
growth [5, 6]. The PEVK region serves as an entropic spring,
while the N2B region improves efficiency of the cardiac cycle
via altered calcium sensitivity [7, 8]. Changes in titin isoform
expression relate primarily to the elastic PEVK, N2B, and
inter-adjacent immunoglobulin (IG) regions and are mediated
by RBM20, the first splice factor related to human heart
disease [9]. Patients with mutations in Rbm20 express more
compliant titin isoforms associated with dilated cardiomyopathy,
fibrosis, and sudden cardiac death [9, 10]. Both, a naturally
occurring RBM20-deficient rat strain and mice carrying a
deletion of the RBM20 RNA recognition motif (RRM), express
similar giant titin isoforms and recapitulate human RBM20
deficiency [2, 11].
In mice, increased diastolic compliance associated
with longer titin isoforms contributes to improved
cardiac function [11]. In patients, the shift in titin
isoformexpression from the stiff N2B to the more compliant
N2BA isoform is also associated with improved function
[4, 12]. This change in titin-based elasticity compensates
for the increased ventricular stiffness by fibrosis. To
evaluate if cardiac splicing could serve as a therapeutic
target to decrease titin-based stiffness and could provide
a lasting beneficial effect on diastolic dysfunction, we
crossed the splice-deficient Rbm20ΔRRM mouse and the
titin N2B knockout mouse (Titin N2B−/−). Excision of
the elastic N2B element of titin affects the mechanical
properties of the sarcomere, hypertrophy signaling, and
ultimately leads to a restrictive filling pattern [5]. In
double-deficient mice (TitinΔN2B/ΔN2B Rbm20ΔRRM/WT),
reduced splicing with expression of more compliant titin
isoforms had several positive effects. Not only was
diastolic compliance improved but also cardiac dimensions,
RNA levels of genes related to the cAMP response, and
oxidative phosphorylation were restored. These findings
suggest that RBM20 could be a therapeutic target in
diastolic dysfunction.
Materials and methods
Animal procedures
Mice were sacrificed by cervical dislocation at 100 to 120 days
of age. The hearts were rapidly excised, washed in PBS, and
dissected into atria, septum, right and left ventricle, and tissues
were snap frozen in liquid nitrogen and stored at −80 °C. Mice
were age and s (...truncated)