In 1980, the World Health Organization task force first sought to define and classify cardiomyopathies. They defined cardiomyopathies as "heart muscle diseases of unknown cause" with three main classifications including: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy [1]. Over the next three decades it became patently obvious that this simple definition was not sufficient to describe the complex heterogeneity of diseases present in the patient population. More robust definitions were necessary for mechanistic links to be established and meaningful therapeutics to be developed. Since then the accepted definition of a cardiomyopathy has evolved and the classifications have greatly expanded. The most recent definition from the American Heart Association Council on Clinical Cardiology states: Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that frequently are genetic. Cardiomyopathies either are confined to the heart or are part of generalized systemic disorders, often leading to cardiovascular death or progressive heart failure–related disability [2]. This latest definition (2006) reflects the growing recognition of molecular genetics as a key factor in the development of cardiomyopathies and highlights the ever-growing complexity of disease classification. Today the genetic basis of HCM and DCM is widely recognized yet our understanding of the precise mechanisms underlying the disease remains unclear. To add to this disconnect, by the time patients become symptomatic, pathology has progressed past the initial phase, where meaningful treatment could occur, to advanced end-stage pathology. By this time often the only treatment options available become "blunt sword" therapeutics that are non-specific and used primarily for symptom management. In fact, over the last 3 decades there has been a marked decline in the innovation of cardiovascular pharmaceuticals owed partially to the vast complexity of disease presentation and progression [3]. In this dissertation, I will focus on a genetic sarcomeric DCM caused by a mutation in alpha-tropomyosin (Tm). Using novel accurate mouse models as a tool we will define the mechanism by which it leads to disease, investigate how disease severity due to the mutation is modified in an age-dependent manner, and examine what this mechanism could mean in the larger picture of cardiomyopathic disease progression. I hope to convince you that by using accurate models of this DCM at multiple levels of biological complexity to tease out the precise mechanisms of disease we can establish meaningful genotype-phenotype relationships that could lead to the development of specific novel therapeutics.

Introduction Russell-Silver Syndrome (RSS) is a genetically determined condition characterized by severe intrauterine and postnatal growth retardation; relative macrocephaly; a small, triangular face; and fifth-finger clinodactyly. The etiology of RSS involves epigenetic regulation through either uniparental disomy or genomic imprinting via DNA methylation. There has been no documented association between RSS and cardiomyopathy. Methods We present an original case of a 32-year-old woman with RSS with dilated a cardiomyopathy who on cardiac biopsy showed occasional hypertrophic and atrophic myocytes with no evidence of inflammation, abnormal sarcomeres and disintegration of the Z bands on ultrastructural analysis, abnormal desmin, and normal C9 immunoreactivity. Conclusion This case represents the first reported association between RSS and cardiomyopathy. Given the complex mechanisms of disease etiology in RSS, this novel case provides insights into the mechanism of progressive dilated cardiomyopathy in an older individual with RSS.

Proper contraction of striated muscle requires the assembly of actin filaments with precise spacing, polarity and lengths, however the mechanisms by which the cell accomplishes this remain unclear. In one model, the giant protein nebulin is proposed to function as a "molecular ruler" specifying the final lengths of the actin filaments. This dissertation focuses on determining the mechanisms by which nebulin regulates thin filament assembly. We found that nebulin physically interacts with CapZ, a protein that caps the barbed end of the actin filament within the Z-disc. Reduction of nebulin levels in chick skeletal myocytes via siRNA results in a reduction of assembled CapZ, and a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin-filament barbed ends to the Z-disc via a direct interaction with CapZ. Unexpectedly, the CapZ binding site was mapped to a site on nebulin that was previously predicted to localize outside of the Z-disc. Thus, we also propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres. To determine the mechanism by which nebulin regulates thin filament length and directly test the molecular ruler hypothesis, a unique small nebulin molecule ("mini-nebulin") was constructed. The introduction of mini-nebulin into chick skeletal myocytes, with endogenous nebulin knocked down, does not result in corresponding shorter actin filaments; an observation that is inconsistent with a strict ruler function. Treatment of these cells, however, with the actin depolymerizing agent Latrunculin A produces filaments that match the length of the mini-nebulin molecule, indicating mini-nebulin stabilizes the actin filaments. Furthermore, knockdown of nebulin results in more dynamic populations of the thin filament components actin, tropomyosin and tropomodulin. Strikingly, introduction of mini-nebulin is able to restore the normal stability of the actin filaments. Taken together, these data indicate that nebulin is responsible for proper actin organization within the Z-disc and contributes to actin filament length regulation by stabilizing the filament, preventing actin depolymerization.

No description available.

Thesis advisor: Eric S. Folker / Thesis advisor: David R. Burgess / During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In many muscle diseases mispositioned myonuclei have been used as a hallmark phenotype of disease. A number of studies over the last decade have started to piece together the cytoskeletal elements that govern these movements. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive nuclear movement. However, it is still not clear how these two pools of microtubule motors become specified. In addition, it is not clear how nuclear position impacts the other defining feature of the muscle cell, which is the highly organized contractile network of sarcomeres. Previously, mispositioned myonuclei have been correlated with improper muscle function, yet no direct link between nuclear position and sarcomere development or function has been demonstrated. In this thesis, we show a role for Aplip1 (the Drosophila homolog of JIP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. Furthermore, we show that a number of sarcomeric proteins, including ZASP, Actin and β-integrin localize to the nucleus prior to being incorporated into the sarcomere, regardless of nuclear position. Finally, we show that the LINC complex is required for nuclear dependent sarcomere assembly and that disruption of nuclear dependent sarcomere assembly or nuclear position resulted in a compromised sarcomeric network. Together, this thesis adds to the mechanisms that are important in positioning nuclei and shows the first direct link between the nucleus and sarcomere assembly. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with Limb-Girdle Muscular Dystrophy Type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), and distal myopathy, four similar adult-onset, progressive, and autosomal dominant muscular dystrophies--collectively called the myotilinopathies. It is not yet known how myotilin mutations cause muscle disease. To investigate myotilin's role in the pathogenesis of muscle disease, I have created and characterized transgenic mice expressing mutant (Thr57Ile) myotilin under the control of the human skeletal alpha-actin promoter. Like LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization, and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. I show that the mutant myotilin protein properly localizes to the Z-disc, and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus (EDL) muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared to littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. To better understand myotilin function, I also created two separate lines of myotilin domain deletion transgenic mice: one expresses a deletion of the N-terminal domain and the second expresses a deletion of the minimal alpha-actinin binding site. Studies in these mice show that 1) the N-terminal domain of myotilin may be required for normal localization to the Z-disc; 2) interaction with alpha-actinin is not required for localization of myotilin to the Z-disc; and 3) deletion of the alpha-actinin binding site causes an aggregation phenotype similar to that of the TgT57I mouse and myotilinopathy patients. In sum, I have established a promising patho-physiological mouse model that unifies the diverse clinical phenotypes of the myotilinopathies. This mouse model promises to be a key resource for understanding myotilin function, unraveling LGMD1A pathogenesis, and investigating therapeutics. / Dissertation

Abstract The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed. Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins. The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.

Background-In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results-Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wildtype mice. No sarcomeric changes were observed in R92Q versus wild-type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E-C coupling and signaling were minimal in E163R myocardium. Conclusions-In E163R models, mutation-driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocytesignaling and E-C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

No description available.

We identified a unique family with autosomal dominant heart disease variably expressed as restrictive cardiomyopathy (RCM), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM), and sought to identify the molecular defect that triggered divergent remodeling pathways. Polymorphic DNA markers for nine sarcomeric genes for DCM and/ or HCM were tested for segregation with disease. Linkage to eight genes was excluded, but a cardiac troponin T (TNNT2) marker cosegregated with the disease phenotype. Sequencing of TNNT2 identified a heterozygous missense mutation resulting in an I79N substitution, inherited by all nine affected family members but by none of the six unaffected relatives. Mutation carriers were diagnosed with RCM (n = 2), non-obstructive HCM (n = 3), DCM (n = 2), mixed cardiomyopathy (n = 1), and mild concentric left ventricular hypertrophy (n = 1). Endomyocardial biopsy in the proband revealed non-specific fibrosis, myocyte hypertrophy, and no myofibrillar disarray. Restrictive Doppler filling patterns, atrial enlargement, and pulmonary hypertension were observed among family members regardless of cardiomyopathy subtype. Mutation of a sarcomeric protein gene can cause RCM, HCM, and DCM within the same family, underscoring the necessity of comprehensive morphological and physiological cardiac assessment in familial cardiomyopathy screening.