Heart failure associated with advanced age is accompanied by molecular and cellular structural remodeling events within cardiomyocytes. One such event is remodeling of the cortical cytoskeleton by actin-binding molecules. Once thought to be a static compartment, the cardiac cytoskeleton is now known to be populated by mechanically- sensitive structural and signaling molecules. This dissertation concerns the role of the mechanosensitive protein vinculin in remodeling of cardiac cortical mechanics and explores its potential role in regulating contractile function with age. Age-related cardiac cytoskeletal reinforcement is shown to be an evolutionarily-conserved mechanism in simians, rats, and Drosophila melanogaster. Mechanistic studies show that cytoskeletal remodeling is correlated with preserved shortening velocity. Finally, cardiac-specific induction of cytoskeletal remodeling through overexpression of vinculin in both juvenile wildtype and myosin heavy chain knockdown flies led to enhanced shortening function and increased hemodynamic stress tolerance. Cytoskeletal reinforcement may play a protective role during aging in multiple cardiac systems by regulating myofilament organization and myocyte shortening

Stem cell differentiation into a variety of lineages is known to involve signaling from the extracellular niche, including from the physical properties of that environment. What regulates stem cell responses to these cues is there ability to activate different mechanotransductive pathways. Here, we will review the structures and pathways that regulate stem cell commitment to a cardiomyocyte lineage, specifically examining proteins within muscle sarcomeres, costameres, and intercalated discs. Proteins within these structures stretch, inducing a change in their phosphorylated state or in their localization to initiate different signals. We will also put these changes in the context of stem cell differentiation into cardiomyocytes, their subsequent formation of the chambered heart, and explore negative signaling that occurs during disease.

An ever-increasing body of literature describes compelling evidence that a subset of young children on the autism spectrum show abnormal cerebral growth trajectories. In these cases, normal cerebral size at birth is followed by a period of abnormal growth and starting in late childhood often by regression compared to unaffected controls. Recent work has demonstrated an abnormal increase in the number of neurons of the prefrontal cortex suggesting that cerebral size increase in autism is driven by excess neuronal production. In addition, some affected children display patches of abnormal laminar positioning of cortical projection neurons. As both cortical projection neuron numbers and their correct layering within the developing cortex requires the undisturbed proliferation of neural progenitors, it appears that neural progenitors lie in the center of the autism pathology associated with early brain overgrowth. Consequently, autism spectrum disorders associated with cerebral enlargement should be viewed as birth defects of an early embryonic origin with profound implications for their early diagnosis, preventive strategies, and therapeutic intervention.

Rationale

Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence.

Objective

Using a highly integrative approach, we sought to determine the molecular trigger of upheld(101) myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction.

Methods and results

Live video imaging of Drosophila cardiac tubes revealed that the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, because of increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with upheld(101) diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and 3-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin-binding sites under low Ca2+ conditions.

Conclusions

As a result of troponin-tropomyosin dysinhibition, upheld(101) hearts exhibited cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.

Psychoactive pharmaceuticals have been found as teratogens at clinical dosage during pregnancy. These pharmaceuticals have also been detected in minute (ppb) concentrations in drinking water in the US, and are environmental contaminants that may be complicit in triggering neurological disorders in genetically susceptible individuals. Previous studies have determined that psychoactive pharmaceuticals (fluoxetine, venlafaxine and carbamazepine) at environmentally relevant concentrations enriched sets of genes regulating development and function of the nervous system in fathead minnows. Altered gene sets were also associated with potential neurological disorders, including autism spectrum disorders (ASD). Subsequent in vitro studies indicated that psychoactive pharmaceuticals altered ASD-associated synaptic protein expression and gene expression in human neuronal cells. However, it is unknown if environmentally relevant concentrations of these pharmaceuticals are able to cross biological barriers from mother to fetus, thus potentially posing risks to nervous system development. The main objective of this study was to test whether psychoactive pharmaceuticals (fluoxetine, venlafaxine, and carbamazepine) administered through the drinking water at environmental concentrations to pregnant mice could reach the brain of the developing embryo by crossing intestinal and placental barriers. We addressed this question by adding (2)H-isotope labeled pharmaceuticals to the drinking water of female mice for 20 days (10 pre-and 10 post-conception days), and quantifying (2)H-isotope enrichment signals in the dam liver and brain of developing embryos using isotope ratio mass spectrometry. Significant levels of (2)H enrichment was detected in the brain of embryos and livers of carbamazepine-treated mice but not in those of control dams, or for fluoxetine or venlafaxine application. These results provide the first evidence that carbamazepine in drinking water and at typical environmental concentrations is transmitted from mother to embryo. Our results, combined with previous evidence that carbamazepine may be associated with ASD in infants, warrant the closer examination of psychoactive pharmaceuticals in drinking water and their potential association with neurodevelopmental disorders.

WD repeat and FYVE domain-containing 3 (WDFY3; also known as Autophagy-Linked FYVE or Alfy) is an identified intellectual disability, developmental delay and autism risk gene. This gene encodes for a scaffolding protein that is expressed in both the developing and adult central nervous system and required for autophagy and aggrephagy with yet unexplored roles in mitophagy. Given that mitochondrial trafficking, dynamics and remodeling have key roles in synaptic plasticity, we tested the role of Wdfy3 on brain bioenergetics by using Wdfy3^+/lacZ mice, the only known Wdfy3 mutant animal model with overt neurodevelopmental anomalies that survive to adulthood. We found that Wdfy3 is required for sustaining brain bioenergetics and morphology via mitophagy. Decreased mitochondrial quality control by conventional mitophagy was partly compensated for by the increased formation of mitochondria-derived vesicles (MDV) targeted to lysosomal degradation (micromitophagy). These observations, extended through proteomic analysis of mitochondria-enriched cortical fractions, showed significant enrichment for pathways associated with mitophagy, mitochondrial transport and axon guidance via semaphorin, Robo, L1cam and Eph-ephrin signaling. Collectively, our findings support a critical role for Wdfy3 in mitochondrial homeostasis with implications for neuron differentiation, neurodevelopment and age-dependent neurodegeneration.

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan.

Heart performance declines with age. Impaired protein quality control (PQC), due to reduced ubiquitin-proteasome system (UPS) activity, autophagic function, and/or chaperone-mediated protein refolding, contributes to cardiac deterioration. The transcription factor FOXO participates in regulating genes involved in PQC, senescence, and numerous other processes. Here, a comprehensive approach, involving molecular genetics, novel assays to probe insect cardiac physiology, and bioinformatics, was utilized to investigate the influence of heart-restricted manipulation of dFOXO expression in the rapidly aging Drosophila melanogaster model. Modest dFOXO overexpression was cardioprotective, ameliorating nonpathological functional decline with age. This was accompanied by increased expression of genes associated predominantly with the UPS, relative to other PQC components, which was validated by a significant decrease in ubiquitinated proteins. RNAi knockdown of UPS candidates accordingly compromised myocardial physiology in young flies. Conversely, excessive dFOXO overexpression or suppression proved detrimental to heart function and/or organismal development. This study highlights D. melanogaster as a model of cardiac aging and FOXO as a tightly regulated mediator of proteostasis and heart performance over time.

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin-linked kinase (ilk) and β1-integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z-bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1-integrin protein levels in old compared with young wild-type flies, and cardiac-specific overexpression of mys in young flies causes aging-like heart dysfunction. Moreover, moderate cardiac-specific knockdown of integrin-linked kinase (ILK)/integrin pathway-associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK-associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine-tuning of this pathway can retard the age-dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin-associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age-dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.