RARG variant predictive of doxorubicin-induced cardiotoxicity identifies a cardioprotective therapy.

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but its use is limited by dose-dependent cardiotoxicity. A recent genome-wide association study identified a SNP (rs2229774) in retinoic acid receptor-γ (RARG) as statistically associated with increased risk of anthracycline-induced cardiotoxicity. Here, we show that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with rs2229774 and who suffered doxorubicin-induced cardiotoxicity (DIC) are more sensitive to doxorubicin. We determine that the mechanism of this RARG variant effect is mediated via suppression of topoisomerase 2ß (TOP2B) expression and activation of the cardioprotective extracellular regulated kinase (ERK) pathway. We use patient-specific hiPSC-CMs as a drug discovery platform, determining that the RARG agonist CD1530 attenuates DIC, and we confirm this cardioprotective effect in an established in vivo mouse model of DIC. This study provides a rationale for clinical prechemotherapy genetic screening for rs2229774 and a foundation for the clinical use of RARG agonist treatment to protect cancer patients from DIC.

Hypertrophic cardiomyopathy ß-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in ß-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

Physiological Mitochondrial Fragmentation Is a Normal Cardiac Adaptation to Increased Energy Demand.

RATIONALE: Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Paradigms have held that mitochondrial fission and fragmentation are the result of pathological stresses, such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in decreased mitochondrial function and cardiac impairment, suggesting that fission is important for maintaining cardiac and mitochondrial bioenergetic homeostasis. OBJECTIVE: The purpose of this study is to determine whether mitochondrial fission and fragmentation can be an adaptive mechanism used by the heart to augment mitochondrial and cardiac function during a normal physiological stress, such as exercise. METHODS AND RESULTS: We demonstrate a novel role for cardiac mitochondrial fission as a normal adaptation to increased energetic demand. During submaximal exercise, physiological mitochondrial fragmentation results in enhanced, rather than impaired, mitochondrial function and is mediated, in part, by ß1-adrenergic receptor signaling. Similar to pathological fragmentation, physiological fragmentation is induced by activation of dynamin-related protein 1; however, unlike pathological fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. Inhibition of fission with P110, Mdivi-1 (mitochondrial division inhibitor), or in mice with cardiac-specific dynamin-related protein 1 ablation significantly decreases exercise capacity. CONCLUSIONS: These findings demonstrate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise, as well as providing additional support for the evolving conceptual framework, where mitochondrial fission and fragmentation play a role in the balance between mitochondrial maintenance of normal physiology and response to disease.

Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling.

The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ß-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.

miR-21 is associated with fibrosis and right ventricular failure.

Combined pulmonary insufficiency (PI) and stenosis (PS) is a common long-term sequela after repair of many forms of congenital heart disease, causing progressive right ventricular (RV) dilation and failure. Little is known of the mechanisms underlying this combination of preload and afterload stressors. We developed a murine model of PI and PS (PI+PS) to identify clinically relevant pathways and biomarkers of disease progression. Diastolic dysfunction was induced (restrictive RV filling, elevated RV end-diastolic pressures) at 1 month after generation of PI+PS and progressed to systolic dysfunction (decreased RV shortening) by 3 months. RV fibrosis progressed from 1 month (4.4% ± 0.4%) to 3 months (9.2% ± 1%), along with TGF-ß signaling and tissue expression of profibrotic miR-21. Although plasma miR-21 was upregulated with diastolic dysfunction, it was downregulated with the onset of systolic dysfunction), correlating with RV fibrosis. Plasma miR-21 in children with PI+PS followed a similar pattern. A model of combined RV volume and pressure overload recapitulates the evolution of RV failure unique to patients with prior RV outflow tract surgery. This progression was characterized by enhanced TGF-ß and miR-21 signaling. miR-21 may serve as a plasma biomarker of RV failure, with decreased expression heralding the need for valve replacement.

iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy.

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-ß) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-ß signalling. TBX20 regulates the expression of TGF-ß signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-ß signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used ß-adrenergic receptor (ß-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), ß2-ARs are a primary source of cAMP/PKA signaling. With longer culture, ß1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic ß-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, ß-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of ß-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

hiPSC Modeling of Inherited Cardiomyopathies.

OPINION STATEMENT: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful new model system to study the basic mechanisms of inherited cardiomyopathies. hiPSC-CMs have been utilized to model several cardiovascular diseases, achieving the most success in the inherited arrhythmias, including long QT and Timothy syndromes (Moretti et al. N Engl J Med. 363:1397-409, 2010; Yazawa et al. Nature. 471:230-4, 2011) and arrhythmogenic right ventricular dysplasia (ARVD) (Ma et al. Eur Heart J. 34:1122-33, 2013). Recently, studies have applied hiPSC-CMs to the study of both dilated (DCM) (Sun et al. Sci Transl Med. 4:130ra47, 2012) and hypertrophic (HCM) cardiomyopathies (Lan et al. Cell Stem Cell. 12:101-13, 2013; Carvajal-Vergara et al. Nature. 465:808-12, 2010), providing new insights into basic mechanisms of disease. However, hiPSC-CMs do not recapitulate many of the structural and functional aspects of mature human cardiomyocytes, instead mirroring an immature - embryonic or fetal - phenotype. Much work remains in order to better understand these differences, as well as to develop methods to induce hiPSC-CMs into a fully mature phenotype. Despite these limitations, hiPSC-CMs represent the best current in vitro correlate of the human heart and an invaluable tool in the search for mechanisms underlying cardiomyopathy and for screening new pharmacologic therapies.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Stabilization of phosphatidylinositol 4-kinase type IIbeta by interaction with Hsp90.

Mammalian cells express two isoforms of type II phosphatidylinositol 4-kinase: PI4KIIα and PI4KIIß. PI4KIIα exists almost exclusively as a constitutively active integral membrane protein because of its palmitoylation (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Südhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). In contrast, PI4KIIß is distributed almost evenly between membranes and cytosol. Whereas the palmitoylated membrane-bound pool is catalytically active, the cytosolic kinase is inactive (Wei, Y. J., Sun, H. Q., Yamamoto, M., Wlodarski, P., Kunii, K., Martinez, M., Barylko, B., Albanesi, J. P., and Yin, H. L. (2002) J. Biol. Chem. 277, 46586-46593; Jung, G., Wang, J., Wlodarski, P., Barylko, B., Binns, D. D., Shu, H., Yin, H. L., and Albanesi, J. P. (2008) Biochem. J. 409, 501-509). In this study, we identify the molecular chaperone Hsp90 as a binding partner of PI4KIIß, but not of PI4KIIα. Geldanamycin (GA), a specific Hsp90 inhibitor, disrupts the Hsp90-PI4KIIß interaction and destabilizes PI4KIIß, reducing its half-life by 40% and increasing its susceptibility to ubiquitylation and proteasomal degradation. Cytosolic PI4KIIß is much more sensitive to GA treatment than is the integrally membrane-associated species. Exposure to GA induces a partial redistribution of PI4KIIß from the cytosol to membranes and, with brief GA treatments, a corresponding increase in cellular phosphatidylinositol 4-kinase activity. Stimuli such as PDGF receptor activation that also induce recruitment of the kinase to membranes disrupt the Hsp90-PI4KIIß interaction to a similar extent as GA treatment. These results support a model wherein Hsp90 interacts predominantly with the cytosolic, inactive pool of PI4KIIß, shielding it from proteolytic degradation but also sequestering it to the cytosol until an extracellular stimulus triggers its translocation to the Golgi or plasma membrane and subsequent activation.

Target identification using drug affinity responsive target stability (DARTS).

Drug Affinity Responsive Target Stability is a general methodology for identifying and studying protein-ligand interactions. The technique is based on the principle that when a small molecule compound binds to a protein, the interaction stabilizes the target protein's structure such that it becomes protease resistant. DARTS is particularly useful for the initial identification of the protein targets of small molecules, but can also be used to validate potential protein-ligand interactions predicted or identified by other means and to estimate the affinity of interactions. The approach is simple and advantageous because it can be performed using crude cell lysates and other complex protein mixtures (without requiring purified proteins), and uses native, unmodified small molecules. The protocols provided in this article describe the general approach for performing DARTS experiments, which can be easily modified and scaled to fit the criteria and purpose of any individual project.

Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase II{alpha}.

Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) and IIbeta (PI4KIIbeta). Nearly all of PI4KIIalpha, and about half of PI4KIIbeta, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIalpha converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIalpha to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIalpha in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIalpha and associates more weakly with lipid rafts.

Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta.

Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.

Structure, function, and regulation of myosin 1C.

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.