Myofibrillar myopathy (MFM), a group of chronic severe muscular disorders, are caused by mutations in genes encoding extramyofibrillar proteins. One of the genes affected and linked with the development of MFM is desmin. The majority of mutations affecting desmin are linked to the development of cardiomyopathy. DES mutations are pleiotropic, generating variable phenotypes, which probably results from interactions between desmin, chaperones and/or other intermediate filaments. However, the factors underlying this variability are not known for the moment. The objective of this work is to investigate the effect of different human desmin heterozygous mutations (S46Y, E245D, D214-E245Del, S419H and E439K) on the function of cardiomyocytes (CMs). For this purpose, induced pluripotent stem cells (hiPSC) were generated from the blood cells of different patients and were successfully differentiated into contractile cardiomyocytes (CMs). Then, cardiomyocytes were cultured in 3D as spheroids to improve the physiological relevance of the model. Using this model, we analyzed and compare their function (contractility, calcium transient, respiration), their gene expression (RNAseq) and their ultrastructure. Our results demonstrated a consistent decrease in mitochondrial respiration in all mutants-CMs compared with control-CMs. However, we observed that all mutant-CMs showed variations in the other functional characteristics, such as calcium transient and contractility. In addition, ultrastructural observations of mitochondria using electron microscopy showed differences in the distribution of mitochondrial subtypes in mutant-CMs compared to control-CMs. The mitochondrial alteration has been validated for one mutation using transcriptomic analysis which showed a lower-expression of genes related to energy metabolism (ATP synthesis and mitochondrial electron transport). Finally, morphological alterations have been also assessed by electronic microscopy and demonstrated several perturbations often related to the degradation or accumulations of myofibrils. Taken together, our results confirm that DES mutations can cause structural and functional defects in human cardiomyocytes. More importantly, these alterations are variable and seem to be correlated to the localization of mutation in DES gene.