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ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery-induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is "energy starved" as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is a complex condition characterized by the inability of the heart to pump sufficient oxygen to the organs to meet their metabolic needs. Among the altered signal transduction pathways associated with HF pathogenesis, the p38 mitogen-activated protein kinase (p38 MAPK) pathway-activated in response to stress- has attracted considerable attention for its potential role in HF progression and cardiac hypertrophy. However, the exact mechanisms by which p38 MAPK influences HF remain unclear. Addressing knowledge gaps may provide insight on why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here we investigate the effects of p38 MAPK inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using a well-established HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed proteomic changes across three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography, while proteome and phosphoproteome profiles were analyzed using multiplexed tandem mass tag labeling and LC-MS/MS. Our findings demonstrate that chronic SB203580 treatment offers protection against progressive decline in cardiac function in HF. The proteomic data indicate that SB203580-treatment exerts broad protection of the cardiac phosphoproteome, beyond inhibiting maladaptive p38-dependent phosphorylation, extending to PKA and AMPK networks among others, ultimately protecting the phosphorylation status of critical myofibrillar and Ca 2+ -handling proteins. Though SB203580 had a more restricted impact on widespread protein changes in HF, its biosignature was consistent with preserved mitochondrial energetics as well as reduced oxidative and inflammatory stress.

Background: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction., Methods: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease., Results: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease., Conclusions: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo., Competing Interests: Disclosures T.A. McKinsey is on the scientific advisory boards of Artemes Bio and Eikonizo Therapeutics, received funding from Italfarmaco for an unrelated project, and has a subcontract from Eikonizo Therapeutics for a small business innovation research grant from the National Institutes of Health (HL154959). The other authors report no conflicts.

Chronic activation of inflammatory pathways (CI) and mitochondrial dysfunction are independently linked to age-related functional decline and early mortality. Interleukin 6 (IL-6) is among the most consistently elevated chronic activation of inflammatory pathways markers, but whether IL-6 plays a causative role in this mitochondrial dysfunction and physical deterioration remains unclear. To characterize the role of IL-6 in age-related mitochondrial dysregulation and physical decline, we have developed an inducible human IL-6 (hIL-6) knock-in mouse (TetO-hIL-6mitoQC) that also contains a mitochondrial-quality control reporter. Six weeks of hIL-6 induction resulted in upregulation of proinflammatory markers, cell proliferation and metabolic pathways, and dysregulated energy utilization. Decreased grip strength, increased falls off the treadmill, and increased frailty index were also observed. Further characterization of skeletal muscles postinduction revealed an increase in mitophagy, downregulation of mitochondrial biogenesis genes, and an overall decrease in total mitochondrial numbers. This study highlights the contribution of IL-6 to mitochondrial dysregulation and supports a causal role of hIL-6 in physical decline and frailty., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models., (© 2022. The Author(s).)

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of >200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

Chronic wounds are a common and debilitating condition associated with aging populations that impact more than 6.5 million patients in the United States. We have previously demonstrated the efficacy of daily topical 1% valsartan in treating wounds in diabetic mouse and pig models. Despite these promising results, there remains a need to develop an extended-release formulation that would reduce patient burden by decreasing the frequency of daily applications. Here, we used nanotechnology to self-assemble valsartan amphiphiles into a filamentous structure (val-filaments) that would serve as a scaffold in wound beds and allow for steady, localised and tunable release of valsartan amphiphiles over 24 days. Two topical treatments of this peptide-based hydrogel on full-thickness wounds in Zucker Diabetic Fatty rats resulted in faster rates of wound closure. By day 23, all val-filament treated wounds were completely closed, as compared to one wound closed in the placebo group. Mechanistically, we observed enrichment of proteins involved in cell adhesion and energetics pathways, downregulation of Tgf-β signalling pathway mediators (pSmad2, pSmad3 and Smad4) and increased mitochondrial metabolic pathway intermediates. This study demonstrates the successful synthesis of a sustained-release valsartan filament hydrogel, its impact on mitochondrial energetics and efficacy in treating diabetic wounds., (© 2021 The Wound Healing Society.)

Although low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we showed that proteomic analyses of human and guinea pig heart failure (HF) were consistent with a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry revealed 32% and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2 mg/kg/d) was sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Although cardiac ATRA declined in guinea pig HF and human IDCM, levels of certain retinoid metabolic enzymes diverged. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, blunted the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys 326 and Lys 328 , are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca 2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys 326 and Lys 328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca 2+ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys 326 and Lys 328 , also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca 2+ -dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca 2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys 328 , modulates muscle contraction via disrupting inhibitory Tpm positioning., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Schmidt et al.)

Purpose: To compare clinical response of intermittent bolus versus continuous infusion neostigmine for acute colonic pseudo-obstruction (ACPO). Acute colonic pseudo-obstruction occurs due to reduced colonic parasympathetic activity. Neostigmine is an acetylcholinesterase inhibitor that increases frequency of smooth muscle contraction by increasing acetylcholine at autonomic nervous system synapses. Although these administration modalities have been studied separately, they have never been compared., Methods: This retrospective study compared bolus versus continuous infusion neostigmine for ACPO. The primary outcome was initial clinical response, defined as bowel movement (BM) within 4 hours of bolus dose or 24 hours of initiation of continuous infusion. Secondary outcomes included time to BM, bowel diameter reduction at 24 hours, incidence of bradycardia, additional neostigmine requirements, and need for colonic decompression or surgical intervention., Results: Seventy-five patients were included (bolus n = 37; infusion n = 38). Median total 24-hour neostigmine dose was 2.0 mg (interquartile range [IQR]: 2.0-2.6) with bolus and 9.6 mg (IQR: 6.3-9.6) with continuous infusion. Initial clinical response was similar (infusion 81.6% vs bolus 62.2%, P = .06), but continuous infusion was associated with greater bowel diameter reduction (73.7% vs 40.5%, P = .004). Bolus administration had shorter time to BM (1.4 vs 3.5 hours, P = .0478) and increased need for colonic decompression (67.6% vs 39.5%, P = .0148). Bolus dosing was associated with less bradycardia (13.5% vs 39.5%, P = 0.011), with no difference in atropine usage (10.8% vs 5.3%, P = .43)., Conclusion: Initial clinical response was similar between groups; however, continuous infusion neostigmine was associated with greater bowel diameter reduction at 24 hours. Bolus administration resulted in less bradycardia; however, given the lack of difference in atropine use, clinical significance is unknown. This study is the first to compare bolus versus continuous infusion neostigmine for ACPO. Further studies are needed to confirm findings.

The renal-outer-medullary‑potassium (ROMK) channel, mutated in Bartter's syndrome, regulates ion exchange in kidney, but its extra-renal functions remain unknown. Additionally, ROMK was postulated to be the pore-forming subunit of the mitochondrial ATP-sensitive K + channel (mitoK ATP ), a mediator of cardioprotection. Using global and cardiomyocyte-specific knockout mice (ROMK-GKO and ROMK-CKO respectively), we characterize the effects of ROMK knockout on mitochondrial ion handling, the response to pharmacological K ATP channel modulators, and ischemia/reperfusion (I/R) injury. Mitochondria from ROMK-GKO hearts exhibited a lower threshold for Ca 2+ -triggered permeability transition pore (mPTP) opening but normal matrix volume changes during oxidative phosphorylation. Isolated perfused ROMK-GKO hearts exhibited impaired functional recovery and increased infarct size when I/R was preceded by an ischemic preconditioning (IPC) protocol. Because ROMK-GKO mice exhibited severe renal defects and cardiac remodeling, we further characterized ROMK-CKO hearts to avoid confounding systemic effects. Mitochondria from ROMK-CKO hearts had unchanged matrix volume responses during oxidative phosphorylation and still swelled upon addition of a mitoK ATP opener, but exhibited a lower threshold for mPTP opening, similar to GKO mitochondria. Nevertheless, I/R induced damage was not exacerbated in ROMK-CKO hearts, either ex vivo or in vivo. Lastly, we examined the response of ROMK-CKO hearts to ex vivo I/R injury with or without IPC and found that IPC still protected these hearts, suggesting that cardiomyocyte ROMK does not participate significantly in the cardioprotective pathway elicited by IPC. Collectively, our findings from these novel strains of mice suggest that cardiomyocyte ROMK is not a central mediator of mitoK ATP function, although it can affect mPTP activation threshold., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function., Methods: To determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties., Results: Reconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity., Conclusions: Our results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

Rationale: Despite increasing prevalence and incidence of heart failure (HF), therapeutic options remain limited. In early stages of HF, sudden cardiac death (SCD) from ventricular arrhythmias claims many lives. Reactive oxygen species (ROS) have been implicated in both arrhythmias and contractile dysfunction. However, little is known about how ROS in specific subcellular compartments contribute to HF or SCD pathophysiology. The role of ROS in chronic proteome remodeling has not been explored., Objective: We will test the hypothesis that elevated mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD., Methods and Results: Using a unique guinea pig model of nonischemic HF that recapitulates important features of human HF, including prolonged QT interval and high incidence of spontaneous arrhythmic SCD, compartment-specific ROS sensors revealed increased mROS in resting and contracting left ventricular myocytes in failing hearts. Importantly, the mitochondrially targeted antioxidant (MitoTEMPO) normalized global cellular ROS. Further, in vivo MitoTEMPO treatment of HF animals prevented and reversed HF, eliminated SCD by decreasing dispersion of repolarization and ventricular arrhythmias, suppressed chronic HF-induced remodeling of the expression proteome, and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that increased mROS in HF disrupts the normal coupling between cytosolic signals and nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca 2+ handling, and action potential repolarization, suggesting new targets for therapeutic intervention., Conclusions: mROS drive both acute emergent events, such as electrical instability responsible for SCD, and those that mediate chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant, and ion transport protein networks. In vivo reduction of mROS prevents and reverses electrical instability, SCD, and HF. Our findings support the feasibility of targeting the mitochondria as a potential new therapy for HF and SCD while identifying new mROS-sensitive protein modifications., (© 2018 American Heart Association, Inc.)

Hypertrophic cardiomyopathy (HCM) stems from mutations in sarcomeric proteins that elicit distinct biophysical sequelae, which in turn may yield radically different intracellular signaling and molecular pathologic profiles. These signaling events remain largely unaddressed by clinical trials that have selected patients based on clinical HCM diagnosis, irrespective of genotype. In this study, we determined how two mouse models of HCM differ, with respect to cellular/mitochondrial function and molecular biosignatures, at an early stage of disease. We show that hearts from young R92W-TnT and R403Q-αMyHC mutation-bearing mice differ in their transcriptome, miRNome, intracellular redox environment, mitochondrial antioxidant defense mechanisms, and susceptibility to mitochondrial permeability transition pore opening. Pathway analysis of mRNA-sequencing data and microRNA profiles indicate that R92W-TnT mutants exhibit a biosignature consistent with activation of profibrotic TGF-β signaling. Our results suggest that the oxidative environment and mitochondrial impairment in young R92W-TnT mice promote activation of TGF-β signaling that foreshadows a pernicious phenotype in young individuals. Of the two mutations, R92W-TnT is more likely to benefit from anti-TGF-β signaling effects conferred by angiotensin receptor blockers and may be responsive to mitochondrial antioxidant strategies in the early stage of disease. Molecular and functional profiling may therefore serve as aids to guide precision therapy for HCM.

Adult stem cells demonstrate metabolic flexibility that is regulated by cell adhesion status. The authors demonstrate that adherent cells primarily utilize glycolysis, whereas suspended cells rely on oxidative phosphorylation for their ATP needs. Akt phosphorylation transduces adhesion-mediated regulation of energy metabolism, by regulating translocation of glucose transporters (GLUT1) to the cell membrane and thus, cellular glucose uptake and glycolysis. Cell dissociation, a pre-requisite for cell transplantation, leads to energetic stress, which is mediated by Akt dephosphorylation, downregulation of glucose uptake, and glycolysis. They designed hydrogels that promote rapid cell adhesion of encapsulated cells, Akt phosphorylation, restore glycolysis, and cellular ATP levels.

Background: We assessed the efficacy and tolerability of 5 low-volume bowel preparations for colonoscopy., Study: We performed an investigator-blinded, randomized trial of 5 bowel preparations: 64 ounces of Gatorade and 306 or 357 g of PEG, both given the day prior; Gatorade and 306 g PEG, 2 L PEG-electrolyte solution with ascorbic acid, and sulfate solution, all 3 given as a split dose. One thousand outpatients consumed their preparation before a morning colonoscopy. The primary endpoint was colon cleanliness assessed by the Chicago Bowel Preparation Scale (BPS). Tolerability was assessed using a subject questionnaire. Another primary endpoint was patient acceptance of a split-dose bowel preparation assessed using a subject questionnaire., Results: No statistically significant differences in the modified Chicago BPS were found among Gatorade and 357 g of PEG given as a day-prior dose and the 3 split-dose arms with 98.5% of colons cleansed adequately. The Gatorade and 357 g of PEG had significantly lower Chicago BPS fluid scores and Chicago BPS total scores (indicating dryer colons that required more irrigation) than the 3 split-dose arms. The Gatorade and PEG preparations were better tolerated. Many subjects are unwilling to consume a split-dose preparation and the majority of subjects would prefer a day-prior preparation with this preference highly dependent on the type of preparation they just consumed., Conclusions: The cleanliness of the colons was not significantly different among the 3 split-dose preparations. Day-prior dosing of Gatorade and 357 g of PEG allowed the mucosa to be visualized as well as did the split-dose preparations.

Here, we examine key regulatory pathways underlying the transition from compensated hypertrophy (HYP) to decompensated heart failure (HF) and sudden cardiac death (SCD) in a guinea pig pressure-overload model by integrated multiome analysis. Relative protein abundances from sham-operated HYP and HF hearts were assessed by iTRAQ LC-MS/MS. Metabolites were quantified by LC-MS/MS or GC-MS. Transcriptome profiles were obtained using mRNA microarrays. The guinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, inflammation, and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, antioxidant enzymes, and other mitochondrial procsses, were downregulated in HF but not HYP. Proteins upregulated in HF implicate extracellular matrix remodeling, cytoskeletal remodeling, and acute phase inflammation markers. Among metabolites, acylcarnitines were downregulated in HYP and fatty acids accumulated in HF. The correlation of transcript and protein changes in HF was weak (R(2) = 0.23), suggesting post-transcriptional gene regulation in HF. Proteome/metabolome integration indicated metabolic bottlenecks in fatty acyl-CoA processing by carnitine palmitoyl transferase (CPT1B) as well as TCA cycle inhibition. On the basis of these findings, we present a model of cardiac decompensation involving impaired nuclear integration of Ca(2+) and cyclic nucleotide signals that are coupled to mitochondrial metabolic and antioxidant defects through the CREB/PGC1α transcriptional axis.

Although the effects of aging and inflammation on the health of the cardiac muscle are well documented, the combined effects of aging and chronic inflammation on cardiac muscle are largely unknown. The renin-angiotensin system (RAS) has been linked independently to both aging and inflammation, but is understudied in the context of their collective effect. Thus, we investigated localized cardiac angiotensin II type I and type II receptors (AT(1)R, AT(2)R), downstream effectors, and phenotypic outcomes using mouse models of the combination of aging and inflammation and compared it to a model of aging and a model of inflammation. We show molecular distinction in the combined effect of aging and inflammation as compared to each independently. The combination maintained an increased AT(1)R:AT(2)R and expression of Nox2 and exhibited the lowest activity of antioxidants. Despite signaling pathway differences, the combined effect shared phenotypic similarities with aging including oxidative damage, fibrosis, and hypertrophy. These phenotypic similarities have dubbed inflammatory conditions as premature aging, but they are, in fact, molecularly distinct. Moreover, treatment with an AT(1)R blocker, losartan, selectively reversed the signaling changes and ameliorated adverse phenotypic effects in the combination of aging and inflammation as well as each independently.

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In striated muscle tropomyosin (Tm) extends along the length of F-actin-containing thin filaments. Its location governs access of myosin binding sites on actin and, hence, force production. Intermolecular electrostatic associations are believed to mediate critical interactions between the proteins. For example, actin residues K326, K328, and R147 were predicted to establish contacts with E181 of Tm. Moreover, K328 also potentially forms direct interactions with E286 of myosin when the motor is strongly bound. Recently, LC-MS/MS analysis of the cardiac acetyl-lysine proteome revealed K326 and K328 of actin were acetylated, a post-translational modification (PTM) that masks the residues' inherent positive charges. Here, we tested the hypothesis that by removing the vital actin charges at residues 326 and 328, the PTM would perturb Tm positioning and/or strong myosin binding as manifested by altered skeletal muscle function and structure in the Drosophila melanogaster model system. Transgenic flies were created that permit tissue-specific expression of K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin and of wild-type actin via the UAS-GAL4 bipartite expression system. Compared to wild-type actin, muscle-restricted expression of mutant actin had a dose-dependent effect on flight ability. Moreover, excessive K328Q and K326Q/K328Q actin overexpression induced indirect flight muscle degeneration, a phenotype consistent with hypercontraction observed in other Drosophila myofibrillar mutants. Based on F-actin-Tm and F-actin-Tm-myosin models and on our physiological data, we conclude that acetylating K326 and K328 of actin alters electrostatic associations with Tm and/or myosin and thereby augments contractile properties. Our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in vivo.

Troponin I (TnI) variant Pro82Ser (cTnIP82S) was initially considered a disease-causing mutation; however, later studies suggested the contrary. We tested the hypothesis of whether a causal link exists between cTnIP82S and cardiac structural and functional remodeling, such as during aging or chronic pressure overload. A cardiac-specific transgenic (Tg) mouse model of cTnIP82S was created to test this hypothesis. During aging, Tg cTnIP82S displayed diastolic dysfunction, characterized by longer isovolumetric relaxation time, and impaired ejection and relaxation time. In young, Tg mice in vivo pressure-volume loops and intact trabecular preparations revealed normal cardiac contractility at baseline. However, upon β-adrenergic stimulation, a blunted contractile reserve and no hastening in left ventricle relaxation were evident in vivo, whereas, in isolated muscles, Ca(2+) transient amplitude isoproterenol dose-response was blunted. In addition, when exposed to chronic pressure overload, Tg mice show exacerbated hypertrophy and decreased contractility compared with age-matched non-Tg littermates. At the molecular level, this mutation significantly impairs myofilament cooperative activation. Importantly, this occurs in the absence of alterations in TnI or myosin-binding protein C phosphorylation. The cTnIP82S variant occurs near a region of interactions with troponin T; therefore, structural changes in this region could explain its meaningful effects on myofilament cooperativity. Our data indicate that cTnIP82S mutation modifies age-dependent diastolic dysfunction and impairs overall contractility after β-adrenergic stimulation or chronic pressure overload. Thus cTnIP82S variant should be regarded as a disease-modifying factor for dysfunction and adverse remodeling with aging and chronic pressure overload., (Copyright © 2015 the American Physiological Society.)

Lysine modifications have been studied extensively in the nucleus, where they play pivotal roles in gene regulation and constitute one of the pillars of epigenetics. In the cytoplasm, they are critical to proteostasis. However, in the last decade we have also witnessed the emergence of mitochondria as a prime locus for post-translational modification (PTM) of lysine thanks, in large measure, to evolving proteomic techniques. Here, we review recent work on evolving set of PTM that arise from the direct reaction of lysine residues with energized metabolic thioester-coenzyme A intermediates, including acetylation, succinylation, malonylation, and glutarylation. We highlight the evolutionary conservation, kinetics, stoichiometry, and cross-talk between members of this emerging family of PTMs. We examine the impact on target protein function and regulation by mitochondrial sirtuins. Finally, we spotlight work in the heart and cardiac mitochondria, and consider the roles acetylation and other newly-found modifications may play in heart disease.

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca(2+)-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+) sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+) concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+) sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+) sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+) sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+) sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+) sensitivity.

Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)-the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1-10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10-275 μM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: Current bowel preparation scales (BPSs) have significant limitations including an inability to distinguish among bowel preparations that adequately cleanse a high percentage of colons. We assessed the reliability and validity of the new Chicago BPS and compared it with existing BPSs., Methods: We performed a prospective evaluation of the cleanliness of 150 colons. Inter-rater agreement was assessed using kappa and Pearson correlation coefficients. Each colon was rated by a gastroenterologist and physician's assistant using the Ottawa BPS, the Boston BPS, a dichotomous (adequate/inadequate) BPS (where adequate was defined as being able to visualize at least 95% of the mucosa), and the Chicago BPS., Results: Pearson correlation coefficients between the gastroenterologists and physician's assistant for total BPS scores were 0.79 (95% confidence interval (CI): 0.73, 0.85), 0.79 (95% CI: 0.72, 0.84), and 0.84 (95% CI: 0.79, 0.88) for the Ottawa, Boston, and Chicago BPSs, respectively. Kappa coefficients for right, middle, and distal colon segment ratings were 0.66, 0.53, and 0.49, respectively, for the Ottawa BPS; 0.64, 0.66, and 0.54, respectively, for the Boston BPS; and 0.70, 0.62, and 0.63, respectively, for the Chicago BPS. Differences between the Chicago BPS and the other BPSs were not statistically significant. The Chicago BPS exhibited the best correspondence between BPS total score and the adequate/inadequate BPS., Conclusions: This study demonstrated the validity and reliability of the Chicago BPS. The better defined grading criteria, better designed numerical ratings scale, and better correspondence between Chicago BPS total score and the adequate/inadequate BPS make the Chicago BPS an attractive alternative to the Ottawa BPS and the Boston BPS.

In the heart, lysine acetylation has been implicated in processes ranging from transcriptional control of pathological remodeling, to cardioprotection arising from caloric restriction. Given the emerging importance of this post-translational modification, we used a proteomic approach to investigate the broader role of lysine acetylation in the heart using a guinea pig model. Briefly, hearts were fractionated into myofilament-, mitochondrial- and cytosol-enriched fractions prior to proteolysis and affinity-enrichment of acetylated peptides. LC-MS/MS analysis identified 1075 acetylated peptides, harboring 994 acetylation sites that map to 240 proteins with a global protein false discovery rate <0.8%. Mitochondrial targets account for 59% of identified proteins and 64% of sites. The majority of the acetyl-proteins are enzymes involved in fatty acid metabolism, oxidative phosphorylation or the TCA cycle. Within the cytosolic fraction, the enzymes of glycolysis, fatty acid synthesis and lipid binding are prominent. Nuclear targets included histones and the transcriptional regulators E1A(p300) and CREB binding protein. Comparison of our dataset with three previous global acetylomic studies uniquely revealed 53 lysine-acetylated proteins. Specifically, newly-identified acetyl-proteins include Ca(2+)-handling proteins, RyR2 and SERCA2, and the myofilament proteins, myosin heavy chain, myosin light chains and subunits of the Troponin complex, among others. These observations were confirmed by anti-acetyl-lysine immunoblotting. In summary, cardiac lysine acetylation may play a role in cardiac substrate selection, bioenergetic performance, and maintenance of redox balance. New sites suggest a host of potential mechanisms by which excitation-contraction coupling may also be modulated.

Rationale: High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce., Objective: To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins., Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values., Conclusions: High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser(23)/Ser(24)) of cardiac troponin (cTn)I results in a decrease in myofilament Ca(2+) sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser(23) and Ser(24) of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser(23) unphosphorylated and Ser(24) phosphorylated, cTnI-DA mimics Ser(23) phosphorylated and Ser(24) unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca(2+) concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca(2+) sensitivity (pCa(50)) was significantly lower in cTnI-DD (pCa(50): 5.39 ± 0.01) compared with cTnI-AA (pCa(50): 5.50 ± 0.01), cTnI-AD (pCa(50): 5.48 ± 0.01), and cTnI-DA (pCa(50): 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa(50) with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca(2+) sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.

Rationale: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery., Objective: To determine the effects of HNO modification in cardiac myofilament proteins., Methods and Results: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively., Conclusions: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

Rationale: Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown., Objective: To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP)., Methods and Results: Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death., Conclusions: The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

Objectives: We assessed the safety, efficacy, and tolerability of a new preparation of Gatorade and polyethylene glycol (PEG) for colonoscopy with or without bisacodyl compared with NuLYTELY., Methods: We performed an investigator-blinded, placebo-controlled, randomized trial of 64 oz of Gatorade and 306 g of PEG (G/PEG) with or without 10 mg of bisacodyl and NuLYTELY. A total of 600 outpatients consumed their preparation the day before a morning colonoscopy. The primary endpoint was colon cleanliness assessed by the Boston Bowel Preparation Scale (BBPS). Tolerability was assessed using a subject questionnaire, and safety was assessed from a basic metabolic profile drawn before the colonoscopy., Results: Adding bisacodyl to G/PEG caused more abdominal bloating/cramps (P<0.01) and did not result in a cleaner colon (P=0.66) compared with G/PEG without bisacodyl. The BBPS scores in both the G/PEG arms and NuLYTELY arm were not significantly different (P=0.19). Compared with subjects in the NuLYTELY arm, subjects in the G/PEG without bisacodyl arm had less nausea (P<0.04), vomiting (P<0.02), abdominal pain (P<0.02), bloating (P<0.005), difficulty drinking the liquid (P<0.0001), and found the overall preparation easier to tolerate (P<0.0001). Subjects in the combined G/PEG arms had a lower serum sodium (P<0.0007), chloride (P<0.007), and BUN (P<0.0001) levels than those in the NuLYTELY arm, but this did not cause any clinical symptoms., Conclusions: Bisacodyl added to G/PEG for colon lavage caused more side effects and did not result in a cleaner colon. The G/PEG preparations cleansed the colon as well as NuLYTELY, were far better tolerated, and were equally safe.

Phosphorylation of mitochondrial proteins has been recognized for decades, and the regulation of pyruvate- and branched-chain α-ketoacid dehydrogenases by an atypical kinase/phosphatase cascade is well established. More recently, the development of new mass spectrometry-based technologies has led to the discovery of many novel phosphorylation sites on a variety of mitochondrial targets. The evidence suggests that the major classes of kinase and several phosphatases may be present at the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix, but many questions remain to be answered as to the location, timing, and reversibility of these phosphorylation events and whether they are functionally relevant. The authors review phosphorylation as a mitochondrial regulatory strategy and highlight its possible role in the pathophysiology of cardiac hypertrophy and failure., (© 2011 Wiley Periodicals, Inc.)

Rationale: Cardiac resynchronization therapy (CRT) is an effective clinical treatment for heart failure patients with conduction delay, impaired contraction, and energetics. Our recent studies have revealed that mitochondrial posttranslational modifications (PTM) may contribute to its benefits, motivating the present study of the oxidative regulation of mitochondrial ATP synthase., Objectives: We tested whether CRT alteration of ATP synthase function is linked to cysteine (Cys) oxidative PTM (Ox-PTM) of specific ATP synthase subunits., Methods and Results: Canine left ventricular myocardium was collected under conditions to preserve Ox-PTM from control, dyssynchronous heart failure (DHF), or hearts that had undergone CRT. In-gel ATPase activity showed that CRT increased ATPase activity by approximately 2-fold (P<0.05) over DHF, approaching control levels, and this effect was recapitulated with a reducing agent. ATP synthase function and 3 Ox-PTM: disulfide bond, S-glutathionylation and S-nitrosation were assessed. ATP synthase from DHF hearts contained 2 novel disulfide bonds, between ATP synthase α subunits themselves and between α and γ subunits, both of which were decreased in CRT hearts (4.38 ± 0.13- and 4.23 ± 0.36-fold, respectively, P<0.01). S-glutathionylation of ATP synthase α subunit occurred in DHF hearts and was decreased by CRT (1.56 ± 0.16-fold, P<0.04). In contrast, S-nitrosation of ATP synthase α subunit in DHF hearts was lower than in CRT hearts (1.53 ± 0.19-fold, P<0.05). All modifications occurred at ATP synthase α subunit Cys294 and Cys to Ser mutation indicated that this residue is critical for ATP synthase function., Conclusions: A selective Cys in ATP synthase α subunit is targeted by multiple Ox-PTM suggesting that this Cys residue may act as a redox sensor modulating ATP synthase function.

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Hypoxia-inducible factor-1alpha (HIF-1α) expression promotes angiogenesis and can influence stem cell engraftment. We investigated the effect of stable over-expression of constitutively active HIF-1α on cardiosphere-derived cell (CDC) engraftment and left ventricular function. CDCs were transduced with a lentivirus expressing a constitutively active mutant of human HIF-1α (LVHIF-1α). Two million male rat CDCs were injected into the infarct following ligation of the mid-LAD in female syngeneic rats. Left ventricular ejection fraction (EF) and circumferential strain were measured by echocardiography at 1 and 4 weeks post-MI in the following groups: PBS group (n = 7), CELL group (n = 7), and CELL-HIF group (n = 7). HIF-1α, VEGF, endothelin-1 expression, and CDC engraftment were measured by quantitative PCR. At 30 days, EF was unchanged in the CELL-HIF group (p = NS), increased in the CELL group (p = 0.025), and decreased in the PBS group (p = 0.021), but engraftment was similar (2.4% ± 3.3% vs 1.7% ± 0.8%, p = NS). Mean circumferential strain of the infarcted region was unchanged in the CELL-HIF group, but improved in the CELL group (p = 0.02). Endothelin-1 and VEGF expression were higher in HIF-CDCs exposed to hypoxia, compared with non-transduced CDCs. HIF-1α expression in CDCs blunted the beneficial functional effects of CDC transplantation, suggesting that paracrine factor balance may play an important role in cardiac regeneration.

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca(2+)-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Rationale: Ca(2+) control of troponin-tropomyosin position on actin regulates cardiac muscle contraction. The inhibitory subunit of troponin, cardiac troponin (cTn)I is primarily responsible for maintaining a tropomyosin conformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of its C-terminal domain (residues 193 to 210) is unclear. Mutations within this region are associated with restrictive cardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning., Objective: We sought to investigate the effect of a cTnI deletion-removal of 17 amino acids from the C terminus- on the structure of troponin-regulated tropomyosin bound to actin., Methods and Results: A truncated form of human cTnI (cTnI(1-192)) was expressed and reconstituted with troponin C and troponin T to form a mutant troponin. Using electron microscopy and 3D image reconstruction, we show that the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca(2+). Specifically, it biases tropomyosin position toward an "enhanced C-state" that exposes more of the myosin-binding site on actin than found with wild-type troponin., Conclusions: In addition to its well-established role of promoting the so-called "blocked-state" or "B-state," cTnI participates in proper stabilization of tropomyosin in the "Ca(2+)-activated state" or "C-state." The last 17 amino acids perform this stabilizing role. The data are consistent with a "fly-casting" model in which the mobile C terminus of cTnI ensures proper conformational switching of troponin-tropomyosin. Loss of actin-sensing function within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient to perturb tropomyosin conformation.

The special blood group antigen Mi.III exhibits a characteristic hybrid structure of glycophorin A (GPA) and glycophorin B, termed Gp.Mur. This phenotype has exceptionally high occurrence rates in several indigenous tribes in Taiwan ( approximately 21.2%-88.4%). Because glycophorin/Miltenberger begins interaction with anion exchanger-1 (AE1) in the endoplasmic reticulum, we hypothesized that the AE1-based macrocomplexes on erythrocyte membranes obtained from Mi.III(+) people could be differentiated from those obtained from non-Miltenberger people. Quantitative mass spectrometric comparison of the AE1-based complexes by iTRAQ (Applied Biosystems) revealed 25% to 67% higher expression of AE1 in Mi.III(+) erythrocytes. In accordance with the higher AE1 level, the Mi.III(+) erythrocytes exhibited superior HCO(3)(-) capacities, pH homeostasis, and osmotic resistance. Cotransfection experiments in HEK293 cells showed that Gp.Mur, like GPA, enhanced trafficking of AE1 to the plasma membrane. In summary, the increased surface expression of AE1 in Mi.III(+) erythrocytes could be attributed to the additive effect of GPA and Gp.Mur coexpression.

As we learn more about the factors that govern cardiac mitochondrial bioenergetics, fission and fusion, as well as the triggers of apoptotic and necrotic cell death, there is growing appreciation that these dynamic processes are finely-tuned by equally dynamic post-translational modification of proteins in and around the mitochondrion. In this minireview, we discuss the evidence that S-nitrosylation, glutathionylation and phosphorylation of mitochondrial proteins have important bioenergetic consequences. A full accounting of these targets, and the functional impact of their modifications, will be necessary to determine the extent to which these processes underlie ischemia/reperfusion injury, cardioprotection by pre/post-conditioning, and the pathogenesis of heart failure.

Spectral and wavelet analyses were performed on ultrasound radiofrequency (RF) data collected from centrifuged cell samples containing HEp-2 cells after induction of apoptosis by exposure to camptothecin. Samples were imaged at several time points after drug exposure using high-frequency ultrasound in the range from 10-60 MHz. A 20-MHz transducer with a f-number of 2.35 and a 40-MHz transducer with a f-number of 3 were used for collecting the RF data. Normalized power spectra were computed from the backscattered ultrasound signals within a region-of-interest (ROI) for further analysis. Spectral slopes, integrated backscatter coefficients (IBCs) and wavelet parameters were estimated as a function of treatment time to monitor acoustic property changes during apoptosis. Changes in spectral parameters were detected starting six hours after treatment and coincided with changes in corresponding histology. Throughout the course of chemotherapy, variation in estimates of the spectral slope of up to 35% were observed. During the treatment, IBCs increased by 400% compared with estimates obtained from the control samples. Changes in spectral parameters are hypothesized to be linked to structural cell changes during apoptosis. In addition, the sensitivity of a wavelet-based analysis to the ultrasonic assessment of cellular changes was investigated. Results of the wavelet analysis showed variations similar to the spectral parameters. Where values of the spectral slope decreased, estimates of the scaling factors increased. Because wavelet analysis preserves the signal-time localization, its application will be potentially beneficial for assessing treatment responses in vivo. The current study contributes toward the development of a non-invasive method for monitoring apoptosis as a measure of the success of chemotherapeutic treatment of cancer.

Congenital long- or short-QT syndrome may lead to life-threatening ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in CAPON, a neuronal nitric oxide synthase (NOS1) regulator, have recently been associated with QT interval variations in a human whole-genome association study. CAPON had been unsuspected of playing a role in cardiac repolarization; indeed, its physiological role in the heart (if any) is unknown. To define the biological effects of CAPON in the heart, we investigated endogenous CAPON protein expression and protein-protein interactions in the heart and performed electrophysiological studies in isolated ventricular myocytes with and without CAPON overexpression. We find that CAPON protein is expressed in the heart and interacts with NOS1 to accelerate cardiac repolarization by inhibition of L-type calcium channel. Our findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.

The subunit composition of the mitochondrial ATP-sensitive K(+)-channel (mitoK(ATP)) is unknown, though some suspect a role for the inward rectifier, Kir6.1, based largely on antibody studies of heart mitochondria. To ascertain the molecular identity of mitoK(ATP) we therefore sought to purify this putative mitochondrial Kir6.1, and conclusively identify the subunits by mass spectrometry. Immunoblots, conducted with two commercially available antibodies, revealed two distinct signals in isolated heart mitochondria, of 51 and 48kDa, respectively. Localization was confirmed by either immuno-gold electron microscopy or by immunofluorescence. Each putative Kir6.1 species was extracted, purified, and identified by LC-MS/MS. The 51kDa band was identified as NADH-dehydrogenase flavoprotein 1, while the preponderant protein in the 48-kDa band was mitochondrial isocitrate dehydrogenase (NADP form). 1D-, 2D-, and native gel analyses were consistent with these assignments. The data suggest it is premature to assign Kir6.1 a role in mitoK(ATP) on the basis of immunoreactivity alone.