Abstract Body (Do not enter title and authors here): Background: Class-5 myosin motor proteins including the members MYO5a, b and c are responsible for intracellular sorting and trafficking of different cargoes, e.g. receptors, metabolites, mRNAs, proteins.

Aim: The aim is to examine the expression and role of class-5 myosins in the heart with specific focus on MYO5b.

Methods: By performing next-generation sequencing, a cohort of SCDY/SIDS patients was screened for variants in MYO5b. Expression analysis were performed using RNA-Seq, qRT-PCR, immunohistochemistry and Western blot. Protein binding partners were identified by proteomics following immunoprecipitation. Mice with a cardiac-specific knockout (KO) of MYO5b (aMHC-Cre^tg/-;MYO5b^flox/flox) and isolated cardiomyocytes were analysed regarding heart function, morphology and molecular alterations compared with controls.

Results: MYO5a and MYO5b encoding the myosin-5a and -5b heavy chains show an expression pattern dependent on the developmental status. A reduction in myosin-5b protein expression was detectable in a cohort of human end-stage failing hearts. Human genetic analysis of a sudden cardiac death in the young/sudden infant death (SCDY/SIDS) cohort (n=95) revealed rare and likely pathogenic missense variants in MYO5b in 3%. Young MYO5b-KO mice showed no difference in heart function compared with WT mice, however, displayed impaired electric conductance, metabolism and reduced sarcomeric integrity. Aging KO mice developed heart failure (HF) ending fatal in all mice. RNA-Seq of left ventricular tissue prior to HF revealed deregulated gene expression of sarcomeric and metabolic genes, ion channel subunits and genes involved in Ca²⁺-homeostasis. Proteomic analysis of myosin-5b binding partners revealed them to be mostly ribosomal and mitochondrial in nature. The mitochondrial membrane potential in cardiomyocytes with reduced MYO5b was downregulated. RNA-Seq of MYO5b-bound ribonucleoprotein particles (RNPs) revealed mRNAs coding for sarcomere structure and function, ion channel subunits and metabolic proteins.

Conclusion: Myosin-5b is involved in the transport of mRNA/ribosome complexes important for sarcomere function, metabolism, Ca²⁺ homeostasis and ion channels. Genetic variants or pathophysiological conditions impairing cardiac MYO5b-related transport are associated with an increased risk for cardiomyocyte dysfunction, HF and sudden death.