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The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain are known to induce cardiomyopathy. The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. We ablated exon3 encoding the first NR of Nebl and created global Neblex3-/ex3- knockout mice. Cardiac function, structure, and transcriptome were assessed before and after a 4-wk treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Neblex3-/ex3- mice demonstrated diastolic dysfunction with preserved systolic function at 6 mo of age. After treadmill running, 4-mo-old Neblex3-/ex3- mice developed concentric cardiac hypertrophy and left ventricular dilation compared with running Nebl+/+ and sedentary Neblex3-/ex3- mice. Disturbance of sarcomeric Z-disks and thin filaments architecture and disruption of intercalated disks and mitochondria were found in exercised Neblex3-/ex3- mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, b-catenin, MLP, a-actinin2, and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-h poststrain and redistributed along with F-actin at 72-h poststrain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. We conclude that Nebl's NR ablation causes disturbed sarcomere, Z-disks, and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to biomechanical stress. NEW & NOTEWORTHY We demonstrate that ablation of first nebulin-repeats of sarcomeric nebulette (Nebl) causes diastolic dysfunction in Neblex3-/ex3- mice. Exercise-induced development of diastolic dysfunction, cardiac hypertrophy and ventricular dilation in knockouts. This was associated with sarcomere disturbance, intercalated disks disruption, and mitochondrial distortion upon stress and altered expression of genes involved in Nebl-based stress network. We demonstrate that G202R and A592 mutations alter actin and desmin expression causing disorganization of desmin filaments upon cyclic strain. [ABSTRACT FROM AUTHOR]

To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 ± 5.1 kPa and a diastolic value of 7.8 ± 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 ± 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function. [ABSTRACT FROM AUTHOR]

The arterial wall contains a significant amount of charged proteoglycans, which are inhomogeneously distributed, with the greatest concentrations in the intimal and medial layers. The hypothesis of this study is that the transmural distribution of proteoglycans plays a significant role in regulating residual stresses in the arterial wall. This hypothesis was first tested theoretically, using the framework of mixture theory for charged hydrated tissues, and then verified experimentally by measuring the opening angle of rat aorta in NaCl solutions of various ionic strengths. A three-dimensional finite element model of aortic ring, using realistic values of the solid matrix shear modulus and proteoglycan fixed-charge density, yielded opening angles and changes with osmolarity comparable to values reported in the literature. Experimentally, the mean opening angle in isotonic saline (300 mosM) was 15 ± 17° and changed to 4 ± 19° and 73 ± 18° under hypertonic (2,000 mosM) and hypotonic (0 mosM) conditions, respectively (n = 16). In addition, the opening angle in isotonic (300 mosM) sucrose, an uncharged molecule, was 60 ± 16° (n = 11), suggesting that the charge effect, not cellular swelling, was the major underlying mechanism for these observations. The extent of changes in opening angle under osmotic challenges suggests that transmural heterogeneity of fixed-charge density plays a crucial role in governing the zero-stress configuration of the aorta. A significant implication of this finding is that arterial wall remodeling in response to altered wall stresses may occur via altered deposition of proteoglycans across the wall thickness, providing a novel mechanism for regulating mechanical homeostasis in vascular tissue. [ABSTRACT FROM AUTHOR]