Michael J. Greenberg

BACKGROUND: Protein quality control is critical for maintaining sarcomere structure and function in cardiomyocytes. Mutations in protein quality control pathway proteins, namely, CRYAB-R120G (crystallin alpha B with arginine to glycine at position 120) and BAG3-P209L (Bcl-2-associated athanogene 3 with proline to lysine at position 209), induce protein aggregates and cardiomyopathy in humans. Novel observations in yeast demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates, and their removal by mitophagy, a lysosomal degradative pathway, facilitates cytosolic protein quality control in cardiomyocytes. METHODS: Mice with inducible cardiac myocyte–specific ablation of TRAF2 (TNF receptor-associated factor 2; TRAF2-icKO [inducible conditional knockout]), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging. Human-induced pluripotent stem cell–derived cardiomyocytes with TRAF2 ablation or R120G knock-in to the CRYAB locus were assessed for protein aggregates and effects of mitophagy stimulation. Transgenic mice expressing R120G-CRYAB protein (R120G-transgenic mice) were subjected to adeno-associated virus 9-cTnT (cardiac troponin T) promoter-driven TRAF2 or PARKIN gain-of-function and TRAF2 loss of function in cardiomyocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. RESULTS: TRAF2-icKO mice demonstrate accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mislocalization to protein aggregates. TRAF2 null human-induced pluripotent stem cell–derived cardiomyocytes demonstrate impaired mitophagy with accumulation of polyubiquitinated proteins and disrupted sarcomeres, which are rescued by both TRAF2 and PARKIN transduction. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic proteins, namely, R120G-CRYAB and P209L-BAG3. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-transgenic mice demonstrate upregulation of myocardial TRAF2 with increased mitophagy. Adult-onset inducible haplo-insufficiency of Traf2 resulted in accelerated mortality, left ventricular systolic dysfunction, and increased protein aggregates in R120G-transgenic mice. Conversely, adeno-associated virus 9-TRAF2 transduction in R120G-transgenic mice stimulated mitophagy, reduced mortality, attenuated LV systolic dysfunction, reduced cytosolic protein aggregates, and restored DESMIN localization. CONCLUSIONS: Stimulation of mitophagy in cardiomyocytes facilitates removal of cytosolic protein aggregates as a mechanism to ameliorate proteotoxic cardiomyopathy.

Iron overload cardiomyopathy (IOC) is caused by elevated systemic iron, and it is characterized by systolic and diastolic dysfunction as well as arrhythmias. Isolating the cardiac-specific cellular and molecular mechanisms driving IOC has been challenging because it affects multiple interconnected organ systems. Here, we leverage stem cells, cardiac tissue engineering, and protein reconstitution to model key contractile aspects of human IOC in vitro and probe the cellular and molecular mechanisms driving cardiac dysfunction. Human-engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced contractile function, impaired relaxation, and increased prevalence of arrhythmogenic events. While both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, cardiomyocytes show higher iron accumulation and reactive oxygen species production. Moreover, iron overload has little effect on the action potential kinetics in engineered heart tissues; however, it impacts the kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and iron-based inhibition of myosin. Our results reveal insights into the cellular and molecular mechanisms of human IOC pathogenesis and establish in vitro models that can be harnessed for mechanistic and translational studies.

Iron overload cardiomyopathy (IOC) is a serious heart condition that is caused by elevated levels of systemic iron. IOC is characterized by both systolic and diastolic dysfunction as well as arrhythmias. It has been challenging to isolate the cardiac-specific cellular and molecular mechanisms driving IOC because the disease affects multiple interconnected organ systems. Here, we leverage stem cell technologies, cardiac tissue engineering, and protein reconstitution assays to model key aspects of human IOC in vitro and to probe the cellular and molecular mechanisms driving cardiac dysfunction. We demonstrate that human engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced systolic function, impaired diastolic function, and increased prevalence of arrhythmogenic events. We demonstrate that while both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, leading to reduced viability, cardiomyocytes show higher levels of iron accumulation and higher levels of reactive oxygen species production. Moreover, we show that in a tissue, iron overload has little effect on the action potential kinetics; however, it directly impacts the amplitude and kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, we demonstrate that iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and direct iron-based inhibition of myosin. In summary, our results reveal new insights into the cellular and molecular mechanisms of human IOC pathogenesis, and they establish new in vitro models that can be harnessed to faithfully recapitulate key aspects of the human disease phenotype. Highlights: Contractile aspects of iron overload cardiomyopathy have been difficult to study in vitro. We developed engineered heart tissues to model key aspects of the human disease. In vitro iron overload reduces contractility and induces arrhythmogenesis. Iron differentially affects cardiomyocytes and cardiac fibroblasts. Iron overload directly impacts the actomyosin contractile apparatus.

Protein arginylation is an essential post-translational modification catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating ribosomal bias and enabling bona fide arginylation identification. The method has been successfully applied to peptide, protein, cell, patient and mouse samples, with 235 unique arginylation sites revealed from human proteomes using 20 µg of input. Representative sites were validated and followed up for their biological functions. This global platform, applicable to various sample types, paves the way for functional studies of this difficult-to-characterize protein modification.

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-y transplant-free survival rate is only ~50 to 70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. Danicamtiv is one of these molecules; however, its direct effects on myosin’s single molecule mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosin’s working stroke without affecting actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically relevant calcium concentrations. We demonstrate important mechanistic differences with another cardiac myosin binding myotrope, omecamtiv mecarbil. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges can contribute to increased cardiac contraction. Taken together, our results have important implications for the design of sarcomeric-targeting compounds for heart failure.

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1’s high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 variant, R256H. We previously identified this variant in a family with dilated cardiomyopathy, who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using cryoelectron microscopy, and we see alterations in actin’s structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1 ^R256H/+ human-induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric organization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.

Acute lower respiratory tract infection (ALRI) remains a major worldwide cause of childhood mortality, compelling innovation in prevention and treatment. Children in Papua New Guinea (PNG) experience profound morbidity from ALRI caused by Streptococcus pneumoniae. As a result of evolutionary divergence, the human PNG population exhibits profound genetic variation and diversity. To address unmet health needs of children in PNG, we tested whether genetic variants increased ALRI morbidity. Whole-exome sequencing of a pilot child cohort identified homozygosity for a novel single-nucleotide variant (SNV) in coenzyme Q6 (COQ6) in cases with ALRI. COQ6 encodes a mitochondrial enzyme essential for biosynthesis of ubiquinone, an electron acceptor in the electron transport chain. A significant association of SNV homozygosity with ALRI was replicated in an independent ALRI cohort (P = 0.036). Mice homozygous for homologous mouse variant Coq6 exhibited increased mortality after pneumococcal lung infection, confirming causality. Bone marrow chimeric mice further revealed that expression of variant Coq6 in recipient (that is, nonhematopoietic) tissues conferred increased mortality. Variant Coq6 maintained ubiquinone biosynthesis, while accelerating metabolic remodeling after pneumococcal challenge. Identification of this COQ6 variant provides a genetic basis for increased pneumonia susceptibility in PNG and establishes a previously unrecognized role for the enzyme COQ6 in regulating inflammatory-mediated metabolic remodeling.

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-year transplant-free survival rate is only ∼50-70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. One such molecule, danicamtiv, recently entered phase II clinical trials; however, its mechanism of action and direct effects on myosin’s mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosin’s working stroke, and in contrast to studies in muscle fibers, we found that it does not affect actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically-relevant calcium concentrations. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges contribute to increased cardiac contraction and improved diastolic function compared to the related myotrope, omecamtiv mecarbil. Taken together, our results have important implications for the design of new sarcomeric-targeting compounds for heart failure. SIGNIFICANCE STATEMENT: Heart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.