SLM2 Splicing Factor Alters Alternative Splicing Pattern in Pressure Overload Mouse Model of Heart Failure

Background:

Previously, we identified Sam68-Like mammalian protein 2 (SLM2) as a novel cardiac splicing regulator with essential functions for maintaining cardiomyocyte integrity by binding to and processing the mRNAs of essential cardiac constituents such as Titin (Ttn). SLM2 was found to be elevated in the failing myocardium, which might be a compensatory mechanism to adapt to the increased wall stress and the hypertrophic response of the heart. Although SLM2's upregulation in dilated cardiomyopathy (DCM) suggests a therapeutic target, particularly in the context of RBM20 cardiomyopathy, its functional roles remain underexplored. This study investigates the impact of SLM2 knockout on cardiac physiology and splicing regulation, aiming to fill the knowledge gap and validate its therapeutic potential.

Methodology:

In order to identify the molecular mechanisms through alternative splicing, we investigated cardiac physiological functions in conditional and heart specific SLM2 deficient mouse model. Cardiac function was assessed through echocardiography, and transcriptomic changes were probed via RNA sequencing to evaluate alternative splicing modifications. To induce cardiac stress and model pressure overload, O-ring aortic banding (ORAB) surgery was performed, allowing us to investigate the stress-adaptive capacity of the heart in the absence of SLM2.

Results:

At eight weeks post-induction, SLM2 KO mice exhibited no significant change in cardiac morphology or function, with echocardiographic parameters comparable to controls. While transcriptomic analysis showed no relevant changes in overall gene expression, it revealed dysregulation in the alternative splicing of sarcomeric genes, suggesting a latent effect of SLM2 depletion. However, a comprehensive GO analysis of alternative splicing events revealed significant (up to 2.5 fold enrichment) splicing alterations in pathways crucial for cardiac muscle tissue development and cellular stress response. Specifically, the KO mice exhibited marked changes (p-val < 0.05) in exon usage in genes central to cardiac function, including differential splicing in titin (Ttn), myosin light chain 2 (Myl2), and troponin T2 (Tnnt2), highlighting the role of SLM2 in splicing regulation. Furthermore, an increased (1.8-fold) expression of the SLM2 family member SAM68 was noted, suggesting an adaptive compensatory response. Stress-induced vulnerability was manifested after ORAB surgery, with KO mice displaying a more pronounced decline in ventricular function and higher mortality rates compared to controls, thus revealing a critical role of SLM2 in cardiac stress. Finally, in order to identify the novel target mRNA transcripts that are SLM2 regulated under this condition, we conducted (RNA immunoprecipitation) RIP experiments with different splicing factors in wild type and SLM2 KO mice 8 weeks after ORAB surgery and the results are currently being analyzed.

Conclusion:

Our preliminary data demonstrates that the alternative splicing patterns of mRNAs display differences in SLM2 knockout mice that have undergone ORAB surgery. In conclusion, SLM2 affects sarcomere gene splicing under stress conditions and SLM2 deficiency leads to cardiac vulnerability in a pressure overload model.