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CSRP3 (cysteine rich protein or muscle LIM protein / MLP) is expressed in striated muscle and localizes, apart from other compartments, to the sarcomeric z-disc and nucleus. CSRP3 acts as a cardiomyocyte mechanical stretch sensor with various mutations in this gene being associated with different cardiomyopathies, among which we previously demonstrated a disease causing role for CSRP3:c. 10T>C, p.W4R, but the underlying molecular mechanisms remain unclear. CSRP3:c.10T>C, p.W4R introduces a novel CpG site, which is prone to methylation and which may lead to effects on transcription, splicing and expression. Long reads mRNA seq performed on Csrp3W4R/W4Rmice revealed the presence of different CSRP3 splice variants with a highly prevalent CSRP3 mRNA missing exon 2 (?2mRNA). Minigene experiments confirmed skipping of exon 2 in the presence of 10T >C. The CpG site formed by the 10T>C is prone to methylation and this event correlates with alternative splicing. Alternative splicing impairs translation of the full length CSRP3 protein and induces translation of a 17KD COOH-terminal isoform from a non-canonical start codon (confirmed by mass spec analysis). The truncated protein localizes to the z-disc where it acts as a toxic peptide and likely induces unfolded protein response. RNA seq of both, human W4R explanted hearts and humanized mouse W4R knock in hearts, revealed significant downregulation of CSRP3. In addition, pathway analysis indicated the significant enrichment of OXPHOS, mitochondrial dysfunction, sirtuin signaling, oxidative stress (Nrf2) and protein misfolding pathways, all of which indicate defects in energy metabolism. Moreover, animals overexpressing ?2mRNA develop heart failure reminiscent of what can be observed in human mutation carriers. In summary, CSRP3:c.10T>C;p.W4R causes cardiomyopathy and associated heart failure via multiple, simultaneous molecular mechanisms, including a splicing defect leading to the expression of novel, pathological COOH-terminal CSRP3 isoforms, loss of CSRP3 mRNA and protein, defects in protein / protein interaction and mislocalization of the mature protein. Therefore, a single mutation may cause disease via multiple mechanisms and hence explains pleiotropy observed in patients.