Utility of common in vitro systems for predicting circulating metabolites.

In vitro systems such as cultured hepatocytes are used early in drug development as a proxy for in vivo data to predict metabolites in human and the potential pre-clinical species. These data support preclinical species selection for toxicology studies as well as provide early evidence for potential active and reactive metabolites that can be generated in human. While in vivo data would be best to select preclinical species for a given compound, only in vitro systems are available when selecting tox species. However, as with any in vitro system, the correlation to actual in vivo results can be variable. Understanding the predictivity of a given in vitro assay for in vivo metabolism would help drug development teams appreciate the significance of early cross-species metabolite profiles relative to the eventual clinical outcomes. In a retrospective analysis of historic metabolite profiling data from Abbott/AbbVie, in vitro systems predicted ~50% of circulating metabolites present in vivo, across preclinical species and human, with no correlation between apparent exposures in vitro vs in vivo A direct comparison of five common in vitro systems using commercial compounds with known metabolism resulted in suspension hepatocytes and co-cultured hepatocytes slightly outperforming the other systems in successfully generating major human circulating metabolites. Current in vitro systems have value early in development when in vivo studies are not feasible and are required for regulatory filings to support pre-clinical toxicology species selection but should not be treated as wholly representative of a given drug's in vivo metabolism. Significance Statement This is a comprehensive assessment of historic metabolism data quantitating the success rate of in vitro to in vivo predictivity. Reliability of in vitro systems for metabolite profiling is important for early drug development, and understanding predictivity will help give appropriate context to the data. New data were also generated to compare common in vitro liver models to determine whether any could be definitively identified as more predictive of human circulating metabolites than others.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Cardioprotection via the skin: nociceptor-induced conditioning against cardiac MI in the NIC of time.

Timely reperfusion is still the most effective approach to limit infarct size in humans. Yet, despite advances in care and reduction in door-to-balloon times, nearly 25% of patients develop heart failure postmyocardial infarction, with its attendant morbidity and mortality. We previously showed that cardioprotection results from a skin incision through the umbilicus in a murine model of myocardial infarction. In the present study, we show that an electrical stimulus or topical capsaicin applied to the skin in the same region induces significantly reduced infarct size in a murine model. We define this class of phenomena as nociceptor-induced conditioning (NIC) based on the peripheral nerve mechanism of initiation. We show that NIC is effective both as a preconditioning and postconditioning remote stimulus, reducing infarct size by 86% and 80%, respectively. NIC is induced via activation of skin C-fiber nerves. Interestingly, the skin region that activates NIC is limited to the anterior of the T9-T10 vertebral region of the abdomen. Cardioprotection after NIC requires the integrity of the spinal cord from the region of stimulation to the thoracic vertebral region of the origin of the cardiac nerves but does not require that the cord be intact in the cervical region. Thus, we show that NIC is a reflex and not a central nervous system-mediated effect. The mechanism involves bradykinin 2 receptor activity and activation of PKC, specifically, PKC-α. The similarity of the neuroanatomy and conservation of the effectors of cardioprotection supports that NIC may be translatable to humans as a nontraumatic and practical adjunct therapy against ischemic disease. NEW & NOTEWORTHY This study shows that an electrical stimulus to skin sensory nerves elicits a very powerful cardioprotection against myocardial infarction. This stimulus works by a neurogenic mechanism similar to that previously elucidated for remote cardioprotection of trauma. Nociceptor-induced conditioning is equally potent when applied before ischemia or at reperfusion and has great potential clinically.

Cardiac inflammation in genetic dilated cardiomyopathy caused by MYBPC3 mutation.

Cardiomyopathies are a leading cause of heart failure and are often caused by mutations in sarcomeric genes, resulting in contractile dysfunction and cellular damage. This may stimulate the production of a robust proinflammatory response. To determine whether myocardial inflammation is associated with cardiac dysfunction in dilated cardiomyopathy (DCM) caused by MYBPC3 mutation, we used the well-characterized cMyBP-C(t/t) mouse model of DCM at 3months of age. Compared to wild type (WT) mice, DCM mice exhibited significantly decreased fractional shortening (36.4±2% vs. 15.5±1.0%, p<0.0001) and significantly increased spleen weight (5.3±0.3 vs. 7.2±0.4mg/mm, p=0.002). Intriguingly, flow cytometry analysis revealed a significant increase in total (CD45+CD11b+Ly6C-MHCII+F480+) macrophages (6.5±1.4% vs. 14.8±1.4%, p=0.002) and classically activated (CD45+CD11b+Ly6C-MHCII+F480+CD206-) proinflammatory (M1) macrophages (3.4±0.8% vs. 10.3±1.2%, p=0.0009) in DCM hearts as compared with WT hearts. These results were further confirmed by immunofluorescence analysis of heart tissue sections. Splenic red pulp (CD11b+Ly6C+MHCIIlowF480hi) macrophages were significantly elevated (1.3±0.1% vs. 2.4±0.1%, p=0.0001) in DCM compared to WT animals. Serum cytokine analysis in DCM animals exhibited a significant increase (0.65±0.2 vs. 2.175±0.5pg/mL, p=0.02) in interleukin (IL)-6 compared to WT animals. Furthermore, RNA-seq analysis revealed the upregulation of inflammatory pathways in the DCM hearts. Together, these data indicate a robust proinflammatory response in DCM hearts, likely in response to cellular damage triggered by MYBPC3 mutation and resultant contractile dysfunction.

N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage/monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage/monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1ß (IL-1ß)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy.

AIMS: A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. METHODS AND RESULTS: To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson's trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E': 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). CONCLUSION: Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.

Cardiac Myosin Binding Protein-C Autoantibodies are Potential Early Indicators of Cardiac Dysfunction and Patient Outcome in Acute Coronary Syndrome.

The degradation and release of cardiac myosin binding protein-C (cMyBP-C) upon cardiac damage may stimulate an inflammatory response and autoantibody (AAb) production. We determined whether the presence of cMyBP-C-AAbs associated with adverse cardiac function in CVD patients. Importantly, cMyBP-C-AAbs were significantly detected in ACS patient sera upon arrival to the emergency department, particularly in STEMI patients. Patients positive for cMyBP-C-AAbs had a reduced LVEF and elevated levels of clinical biomarkers of MI. We conclude that cMyBP-C-AAbs may serve as early predictive indicators of deteriorating cardiac function and patient outcome in ACS patients prior to the infarction.

High-Throughput Diagnostic Assay for a Highly Prevalent Cardiomyopathy-Associated MYBPC3 Variant.

A 25-basepair deletion variant of MYBPC3 occurs at high frequency in individuals of South Asian descent and is estimated to affect 55 million people worldwide, carrying an increased likelihood of cardiomyopathy. Since this variant is prevalent and severe in this subpopulation, quick and affordable screening to provide risk-assessment to guide treatment for these patients is critical. An RNaseH qPCR assay was developed to quickly and specifically diagnose the presence of the 25-basepair deletion variant in MYBPC3. RNAseH-blocked nucleotide primers were designed to identify the presence or absence of the wild type MYBPC3 allele or the genomic sequence containing the 25-basepair deletion. Using this assay, three blinded operators were able to accurately determine the genotype from human genomic DNA samples from blood and saliva using a qPCR thermocycler. Furthermore, positive variant subjects were examined by both electrocardiography and echocardiography for the presence of cardiomyopathy. A simple, robust assay was established, verified and validated that can be automated to detect the presence of the highly prevalent 25-basepair deletion MYBPC3 variant using both blood and saliva samples. The assay will provide quick and accurate prescreening of individuals at high risk for cardiomyopathies and allow for better clinical identification of 25-basepair deletion MYBPC3 carriers in large cohort epidemiological studies.

Alterations in Multi-Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein-C.

BACKGROUND: The geometric organization of myocytes in the ventricular wall comprises the structural underpinnings of cardiac mechanical function. Cardiac myosin binding protein-C (MYBPC3) is a sarcomeric protein, for which phosphorylation modulates myofilament binding, sarcomere morphology, and myocyte alignment in the ventricular wall. To elucidate the mechanisms by which MYBPC3 phospho-regulation affects cardiac tissue organization, we studied ventricular myoarchitecture using generalized Q-space imaging (GQI). GQI assessed geometric phenotype in excised hearts that had undergone transgenic (TG) modification of phospho-regulatory serine sites to nonphosphorylatable alanines (MYBPC3(AllP-/(t/t))) or phospho-mimetic aspartic acids (MYBPC3(AllP+/(t/t))). METHODS AND RESULTS: Myoarchitecture in the wild-type (MYBPC3(WT)) left-ventricle (LV) varied with transmural position, with helix angles ranging from -90/+90 degrees and contiguous circular orientation from the LV mid-myocardium to the right ventricle (RV). Whereas MYBPC3(AllP+/(t/t)) hearts were not architecturally distinct from MYBPC3(WT), MYBPC3(AllP-/(t/t)) hearts demonstrated a significant reduction in LV transmural helicity. Null MYBPC3((t/t)) hearts, as constituted by a truncated MYBPC3 protein, demonstrated global architectural disarray and loss in helicity. Electron microscopy was performed to correlate the observed macroscopic architectural changes with sarcomere ultrastructure and demonstrated that impaired phosphorylation of MYBPC3 resulted in modifications of the sarcomere aspect ratio and shear angle. The mechanical effect of helicity loss was assessed through a geometric model relating cardiac work to ejection fraction, confirming the mechanical impairments observed with echocardiography. CONCLUSIONS: We conclude that phosphorylation of MYBPC3 contributes to the genesis of ventricular wall geometry, linking myofilament biology with multiscale cardiac mechanics and myoarchitecture.

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation.

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C((t/t))) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.

Surviving the infarct: A profile of cardiac myosin binding protein-C pathogenicity, diagnostic utility, and proteomics in the ischemic myocardium.

Cardiac myosin binding protein-C (cMyBP-C) is a regulatory protein of the contractile apparatus within the cardiac sarcomere. Ischemic injury to the heart during myocardial infarction (MI) results in the cleavage of cMyBP-C in a phosphorylation-dependent manner and release of an N-terminal fragment (C0C1f) into the circulation. C0C1f has been shown to be pathogenic within cardiac tissue, leading to the development of heart failure. Based on its high levels and early release into the circulation post-MI, C0C1f may serve as a novel biomarker for diagnosing MI more effectively than current clinically used biomarkers. Over time, circulating C0C1f could trigger an autoimmune response leading to myocarditis and progressive cardiac dysfunction. Given the importance of cMyBP-C phosphorylation state in the context of proteolytic cleavage and release into the circulation post-MI, understanding the posttranslational modifications (PTMs) of cMyBP-C would help in further elucidating the role of this protein in health and disease. Accordingly, recent studies have implemented the latest proteomics approaches to define the PTMs of cMyBP-C. The use of such proteomics assays may provide accurate quantitation of the levels of cMyBP-C in the circulation following MI, which could, in turn, demonstrate the efficacy of using plasma cMyBP-C as a cardiac-specific early biomarker of MI. In this review, we define the pathogenic and potential immunogenic effects of C0C1f on cardiac function in the post-MI heart. We also discuss the most advanced proteomics approaches now used to determine cMyBP-C PTMs with the aim of validating C0C1f as an early biomarker of MI.