Human mutations that truncate the massive sarcomere protein titin (TTNtv) are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtv, diminish contractile performance and are pathogenic. By combining functional analyses with RNAseq, we explain why truncations in the A-band domain of TTN cause DCM while truncations in the I-band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS-cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodelling.

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, temporal-spatial developmental programs, we performed single-cell RNA sequencing of >1200 murine cells isolated at seven time points spanning E9.5 (primordial heart tube) to P21 (mature heart). Using unbiased transcriptional data we classified cardiomyocytes (CM), endothelial cells (EC), and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. Harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem cell-derived CMs and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatial-temporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease.

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.

BCS1L mutations cause disease phenotypes ranging from highly restricted pili torti and sensorineural hearing loss (the Björnstad syndrome) to profound multisystem organ failure (complex III deficiency and the GRACILE syndrome). All BCS1L mutations disrupted the assembly of mitochondrial respirasomes (the basic unit for respiration in human mitochondria), but the clinical expression of the mutations was correlated with the production of reactive oxygen species. Mutations that cause the Björnstad syndrome illustrate the exquisite sensitivity of ear and hair tissues to mitochondrial function, particularly to the production of reactive oxygen species.