Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

In the current study, the three-dimensional (3D) topologies of dyadic clefts and associated membrane organelles were mapped in mouse ventricular myocardium using electron tomography. The morphological details and the distribution of membrane systems, including transverse tubules (T-tubules), junctional sarcoplasmic reticulum (SR) and vicinal mitochondria, were determined and presumed to be crucial for controlling cardiac Ca2+ dynamics. The geometric complexity of T-tubules that varied in diameter with frequent branching was clarified. Dyadic clefts were intricately shaped and remarkably small (average 4.39×105 nm3, median 2.81×105 nm3). Although a dyadic cleft of average size could hold maximum 43 ryanodine receptor (RyR) tetramers, more than one-third of clefts were smaller than the size that is able to package as many as 15 RyR tetramers. The dyadic clefts were also adjacent to one another (average end-to-end distance to the nearest dyadic cleft, 19.9 nm) and were distributed irregularly along T-tubule branches. Electron-dense structures that linked membrane organelles were frequently observed between mitochondrial outer membranes and SR or T-tubules. We, thus, propose that the topology of dyadic clefts and the neighboring cellular micro-architecture are the major determinants of the local control of Ca2+ in the heart, including the establishment of the quantal nature of SR Ca2+ releases (e.g. Ca2+ sparks).

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Hyperpolarization-activated cyclic nucleotide-gated channel 4 gene HCN4 is a pacemaker channel that plays a key role in automaticity of sinus node in the heart, and an HCN4 mutation was reported in a patient with sinus node dysfunction. Expression of HCN4 in the heart is, however, not confined to the sinus node cells but is found in other tissues, including cells of the conduction system. On the other hand, mutations in another cardiac ion channel gene, SCN5A, also cause sinus node dysfunction as well as other cardiac arrhythmias, including long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, and progressive cardiac conduction disturbance. These observations imply that HCN4 abnormalities may be involved in the pathogenesis of various arrhythmias, similar to the SCN5A mutations. In this study, we analyzed patients suffering from sinus node dysfunction, progressive cardiac conduction disease, and idiopathic ventricular fibrillation for mutations in HCN4. A missense mutation, D553N, was found in a patient with sinus node dysfunction who showed recurrent syncope, QT prolongation in electrocardiogram, and polymorphic ventricular tachycardia, torsade de pointes. In vitro functional study of the D553N mutation showed a reduced membranous expression associated with decreased If currents because of a trafficking defect of the HCN4 channel in a dominant-negative manner. These data suggest that the loss of function of HCN4 is associated with sinus nodal dysfunction and that a consequence of pacemaker channel abnormality might underlie clinical features of QT prolongation and polymorphic ventricular tachycardia developed under certain conditions.

This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

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RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20S637A mouse as a good model for in vivo study.

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