Adriana S. Trujillo

Huntington's Disease Induced Cardiac Amyloidosis Is Reversed by Modulating Protein Folding and Oxidative Stress Pathways in the Drosophila Heart

Amyloid-like inclusions have been associated with Huntingtons disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.

Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart.

Cardiac vinculin overexpression is a conserved aging response that is associated with enhanced myocardial performance and extended organismal life span. Sending in vinculin reinforcements A common charge for graceful aging is to stay “young at heart.” With age, the heart undergoes necessary remodeling to keep it functioning—or young—even though the heart experiences relatively little regeneration in the human lifetime. The mechanisms of remodeling in mammals remain unclear but, if known, could help develop new therapies to treat heart failure, a leading killer in the developed world. Kaushik et al. therefore performed a proteomic analysis in old and young monkeys and rats, and identified one protein at the heart of it all: vinculin. Vinculin is conserved across species, being present at cell-matrix and cell-cell adhesions and also anchoring the cardiomyocyte membrane to its actin cytoskeleton. Thus, Kaushik et al. hypothesized that vinculin accumulates with age to regulate cytoskeletal stiffening and heart cell contractility. This mechanism was confirmed in rats and in different strains of Drosophila, supporting the notion that particular aspects of heart remodeling are beneficial and prolong life span, rather than being maladaptive. By using several models and producing a large proteomic network centered on vinculin and other cytoskeletal proteins, the authors have put forth a valuable resource for better understanding cardiac aging and for selecting therapeutic targets to prevent heart failure and also keep the heart young and beating as we age. The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age‐dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A‐type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age‐dependent heart defects are not well understood. To address this issue, we modeled human disease‐causing mutations in the Drosophila melanogaster Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Genetic analyses demonstrated that simultaneous over‐expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.

Drosophila as a potential model to ameliorate mutant Huntington-mediated cardiac amyloidosis

Several human diseases, including Huntingtons disease (HD), are associated with the expression of mutated, misfolded, and aggregation-prone amyloid proteins. Cardiac disease is the second leading cause of death in HD, which has been mainly studied as a neurodegenerative disease that is caused by expanded polyglutamine repeats in the huntingtin protein. Since the mechanistic basis of mutant HD-induced cardiomyopathy is unknown, we established a Drosophila heart model that exhibited amyloid aggregate-induced oxidative stress, resulting in myofibrillar disorganization and physiological defects upon expression of HD-causing PolyQ expression in cardiomyocytes. Using powerful Drosophila genetic techniques, we suppressed mutant HD-induced cardiomyopathy by modulating pathways associated with folding defects and oxidative stress. In this addendum, we describe additional potential molecular players that might be associated with HD cardiac amyloidosis. Drosophila, with its high degree of conservation to the human genome and many techniques to manipulate its gene expression, will be an excellent model for the suppression of cardiac amyloidosis linked to other polyglutamine expansion repeat disorders.