Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction

Abstract

Background Left Ventricular (LV) stiffening contributes to Heart Failure with preserved Ejection Fraction (HFpEF), a syndrome with no effective treatment options. Increasing titin’s compliance in the heart has become possible recently through inhibition of the splicing factor RBM20. Here we investigated the effects of increasing titin’s compliance in mice with diastolic dysfunction. Methods Mice in which the RNA recognition motif (RRM) of one of the RBM20 alleles was floxed and which expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation (TAC/DOCA). RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene) with DMSO injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated using echocardiography and pressure volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with Agarose gels. Results cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, named super compliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ~45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix (ECM) stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCA cRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. Conclusions Inhibition of the RBM20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for HFpEF.

Could Modification of Titin Contribute to an Answer for Heart Failure With Preserved Ejection Fraction?

“…A recent opinion paper by Shah et al (3) discussed phenotypic diversity in the HFpEF population and proposed an approach to treatment based on specific aspects of pathophysiology present in individual patients. The accompanying paper by Methawasin et al (4), with its focus on titin, is an example of how such an approach might ultimately work.…”

“…Thus, titin represents a novel and attractive target for treatment of HFpEF. For readers not familiar with titin, understanding the ground-breaking work of Methawasin and co-workers (4) requires a brief introduction to this important protein.…”

“…Methawasin et al (4) are to be congratulated for a tour de force proof of principle investigation of the effects of manipulation of titin isoforms as a treatment for a transverse aortic constriction (TAC) – DOCA murine model of progressive LV hypertrophy leading to HFpEF. Conditional expression of a transgene with deletion of the RNA recognition motif for one of the RBM20 alleles ( cRbm20 ∆RRM ) resulted in reduced splicing and a substantial increase in larger, more compliant titins.…”

“…This approach is not necessarily futile, as there may be other means to achieve this goal, for example, inhaled or oral nitrites. However, it is important to reiterate that Methawasin et al (4) targeted titin by engineering an isoform switch which, while markedly improving myocardial stiffness and normalizing cardiac function and exercise tolerance, did not reverse an abnormality found in HFpEF. Thus, this promising approach is not dependent on reversing a pre-existing titin abnormality and has the added benefit of speeding relaxation.…”

Response by Methawasin and Granzier to Letter Regarding Article, “Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function in a Mouse Model of Heart Failure With Preserved Ejection Fraction”

“…Lewis and Miller for discussing important questions regarding the possible benefits of Rbm20 inhibition in patients with diastolic dysfunction and, importantly, how to identify those patients who will benefit from titin modification vs. antifibrotic therapies. As shown in two recent studies 1, 2 targeting the titin splicing factor Rbm20 can lessen diastolic dysfunction. For example, Methawasin et al showed that inhibiting the expression of Rbm20 in a mouse model with an HFpEF-like condition upregulates super compliant titin isoforms and improves diastolic function and exercise tolerance, despite persistent fibrosis 1 .…”