Introduction: Unconventional motor proteins myosin (Myo) are involved in intracellular cargo transport of organelles, vesicles, receptors and ion channels along actin filaments. Thereby, they regulate energy substrate transport, receptor signalling, gene expression and ion channel distribution. Here, we analysed the regulation and roles of Myo5a/b during cardiomyocyte development and in physiological and pathophysiological stress responses. 

Methods & results: qRT-PCR analysis revealed a regulation of Myo5a/b dependent on the state of development with Myo5a being the predominant neonatal and 5b the predominant adult isoform in the heart. Physiological stress induced by pregnancy upregulated both isoforms in wildtype (WT) mice (5a +73%, 5b +77%, p<0.05). Additionally, both were regulated contrariwise 3/7 days (d) post myocardial infarction (MI) in WT male mice with Myo5a up- (3d +122%, p<0.05; 7d +76%, p<0.01) and Myo5b downregulated (3d -58%; 7d -39%, p<0.01). Human end-stage failing hearts revealed reduced MYO5B protein levels (-28 %, p<0.05) compared with non-failings. The role of Myo5b was further analysed in the adult heart by generating a cardiomyocyte-specific knockout mouse (KO: αMHC-Cre;Myo5b^flox/flox; WT: Myo5b^flox/flox). Male and female KO mice developed without any obvious phenotypical alterations until 3 months (M) of age, with normal left ventricular (LV) function. However, HOLTER analyses revealed supraventricular extras systoles and atrial fibrillation. With increasing age, KO mice of both sexes gradually developed systolic cardiac dysfunction (6M male: WT 50±15, KO 27±12 %FAC, p<0.001), accompanied by increased fibrosis, inflammation and cardiomyocyte hypertrophy with premature mortality (none of the KO mice survived more than 37 weeks). Pregnancy promoted an earlier onset of this phenotype in female KO mice. RNAseq analysis of LV tissue from 3M old male mice revealed 796 altered genes between KO and WT mice (p-adj. <0.05) and bioinformatical analyses (DAVID tool) showed alterations in several metabolic pathways and in sarcomeric gene expression. Furthermore, reduced cardiac triglyceride content (-49%, p<0.01) and decreased basal (-28%, p<0.05) and insulin-stimulated (-40%, p<0.001) glucose uptake in isolated adult cardiomyocytes (ACM) from KO compared with WT mice confirmed an important role of Myo5b for insulin-dependent glucose uptake and fatty acid oxidation. Further analyses showed that the nuclear mRNA content of sarcomeric genes, i.e. MHC, Tnni3 and Actn2 was not altered while the cytoplasmatic mRNA content was reduced in KO compared with WT ACM. Immunoprecipitation experiments revealed that Myo5b is associated with functional ribosomes in WT hearts.

Conclusion: Myo5a (foetal form, upregulated in pregnancy and after MI) and Myo5b (adult form, upregulated in pregnancy and reduced after MI) display a similar contrarywise regulation during development and in response to pathophysiological stress as sarcomeric genes α- and β-MHC, while the regulation in pregnancy is different. Cardiomyocyte-specific deletion of Myo5b leads to supraventricular extras systoles and atrial fibrillation, heart failure and premature death, features that are accelerated by pregnancy in female mice. Mechanistically, Myo5b is involved in the energy substrate regulation and gene expression at least in part by regulating the transport of mRNA/ribosome complexes.