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.

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness ‘myosinopathy’ patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.

Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57. In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool.

Background: Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity allowing acquisition of contractile or synthetic states but critical information is missing about the physiological signals, promoting formation and maintenance of contractile VSMCs in vivo . BMP9 and BMP10 are known to regulate endothelial quiescence after secretion from the liver and right atrium, whereas a direct role in the regulation of VSMCs was not investigated. Here, we studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs. Methods: We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiological role of BMP9, BMP10, ALK1 and SMAD7 in vivo . Morphometric assessments, expression analysis, blood pressure measurements, single molecule fluorescence in situ hybridization (FISH) were performed together with analysis of isolated pulmonary VSMCs to unravel phenotypic and transcriptomic changes in response to absence or presence of BMP9 and BMP10. Results: Concomitant genetic inactivation of Bmp9 in the germ line and Bmp10 in the right atrium led to dramatic changes in vascular tone and diminution of the VSMC layer with attenuated contractility and decreased systemic as well as right ventricular systolic pressure (RVSP). Vice versa , overexpression of Bmp10 in endothelial cells (ECs) of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as RVSP. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. SMC specific overexpression of Smad7 completely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adult Bmp9/10 double mutants. Deletion of Alk1 in VSMCs recapitulated the Bmp9/10 phenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-FISH uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in different Alk1 mutant vessel beds. Conclusions: Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. Surprisingly, the effects of BMP9/10 in VSMCs are mediated by different combinations of BMP type 1 receptors in a vessel bed specific manner, offering new opportunities to manipulate blood pressure in the pulmonary circulation.

This report describes a dog with recurrent atrial fibrillation and atrial flutter associated with chronic gastritis. Although no underlying structural heart disease was apparent antemortem or on gross post-mortem examination, chronic atrial myocarditis was seen on histopathological examination. Atrial myocarditis is a recognised cause of atrial fibrillation in human beings with presumed lone fibrillation, and an association between supraventricular tachyarrhythmias and infectious agents has been made.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory SMAD6 in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when SMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs prior to their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. SMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPC as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

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