Postprandial cardiac hypertrophy is sustained by mechanics, epigenetic, and metabolic reprogramming in pythons.

Constricting pythons, known for their ability to consume infrequent, massive meals, exhibit rapid and reversible cardiac hypertrophy following feeding. Our primary goal was to investigate how python hearts achieve this adaptive response after feeding. Isolated myofibrils increased force after feeding without changes in sarcomere ultrastructure and without increasing energy cost. Ca2+ transients were prolonged after feeding with no changes in myofibril Ca2+ sensitivity. Feeding reduced titin-based tension, resulting in decreased cardiac tissue stiffness. Feeding also reduced the activity of sirtuins, a metabolically linked class of histone deacetylases, and increased chromatin accessibility. Transcription factor enrichment analysis on transposase-accessible chromatin with sequencing revealed the prominent role of transcription factors Yin Yang1 and NRF1 in postfeeding cardiac adaptation. Gene expression also changed with the enrichment of translation and metabolism. Finally, metabolomics analysis and adenosine triphosphate production demonstrated that cardiac adaptation after feeding not only increased energy demand but also energy production. These findings have broad implications for our understanding of cardiac adaptation across species and hold promise for the development of innovative approaches to address cardiovascular diseases.

Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

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.

Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

Considering impact of age and sex on cardiac cytoskeletal components.

The cardiac cytoskeletal components are integral to cardiomyocyte function and are responsible for contraction, sustaining cell structure, and providing scaffolding to direct signaling. Cytoskeletal components have been implicated in cardiac pathology; however, less attention has been paid to age-related modifications of cardiac cytoskeletal components and how these contribute to dysfunction with increased age. Moreover, significant sex differences in cardiac aging have been identified, but we still lack a complete understanding to the mechanisms behind these differences. This review summarizes what is known about how key cardiomyocyte cytoskeletal components are modified because of age, as well as reported sex-specific differences. Thorough consideration of both age and sex as integral players in cytoskeletal function may reveal potential avenues for more personalized therapeutics.

The influence of estrogen on myocardial post-translational modifications and cardiac function in women.

The lifetime risk of heart failure (HF) is comparable in men and women; nevertheless, disparities exist in our understanding of how HF differs between sexes. Several differences in cardiac physiology exist between men and women including the propensity to develop specific HF phenotypes. Men are more likely to be diagnosed with HF failure with reduced ejection fraction, while women have a greater propensity to develop HF with preserved ejection fraction. The mechanisms responsible for these differences remain unclear. Post-translational modifications (PTMs) of myofilament proteins likely contribute to these sex-specific propensities. The role of PTMs in heart disease is an expanding field with immense potential therapeutic targets. However, numerous PTMs remain underexplored, particularly in the context of the female heart. Estrogen, a key gonadal hormone, cardioprotective in pre-menopausal women and its loss with menopause likely contributes to disease in aging women. However, how estrogen regulates PTMs to contribute to HF development is not fully clear. This review outlines key sex differences in HF along with characterizing the contributions of novel myocardial PTMs in cardiac physiology and their regulation by estrogen. Collectively, we highlight the necessity for further investigation into women's heart health and the distinctive mechanisms distinguishing women from men.

Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.

BACKGROUND: Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive. METHODS: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart. RESULTS: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA. CONCLUSIONS: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.

TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction.

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling.

BACKGROUND: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. METHODS: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. RESULTS: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. CONCLUSIONS: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Serum response factor deletion 5 regulates phospholamban phosphorylation and calcium uptake.

AIMS: Pediatric dilated cardiomyopathy (pDCM) is characterized by unique age-dependent molecular mechanisms that include myocellular responses to therapy. We previously showed that pDCM, but not adult DCM patients respond to phosphodiesterase 3 inhibitors (PDE3i) by increasing levels of the second messenger cAMP and consequent phosphorylation of phospholamban (PLN). However, the molecular mechanisms involved in the differential pediatric and adult response to PDE3i are not clear. METHODS AND RESULTS: Quantification of serum response factor (SRF) isoforms from the left ventricle of explanted hearts showed that PDE3i treatment affects expression of SRF isoforms in pDCM hearts. An SRF isoform lacking exon 5 (SRFdel5) was highly expressed in the hearts of pediatric, but not adult DCM patients treated with PDE3i. To determine the functional consequence of expression of SRFdel5, we overexpressed full length SRF or SRFdel5 in cultured cardiomyocytes with and without adrenergic stimulation. Compared to a control adenovirus, expression of SRFdel5 increased phosphorylation of PLN, negatively affected expression of the phosphatase that promotes dephosphorylation of PLN (PP2Cε), and promoted faster calcium reuptake, whereas expression of full length SRF attenuated calcium reuptake through blunted phosphorylation of PLN. CONCLUSIONS: Taken together, these data indicate that expression of SRFdel5 in pDCM hearts in response to PDE3i contributes to improved function through regulating PLN phosphorylation and thereby calcium reuptake.

Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy.

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

Site-specific acetyl-mimetic modification of cardiac troponin I modulates myofilament relaxation and calcium sensitivity.

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.

From pediatrics to geriatrics: Mechanisms of heart failure across the life-course.

Heart failure (HF) is a significant public health problem and a disease with high 5-year mortality. Although age is the primary risk factor for development of HF, it is a disease which impacts patients of all ages. Historically, HF has been studied as a one-size fits all strategy- with the majority of both clinical and basic science investigations employing adult male subjects or adult male pre-clinical animal models. We postulate that inclusion of biological variables in HF studies is necessary to improve our understanding of mechanisms of HF and improve outcomes. In this review, we will discuss age-specific differences in HF patients, particularly focusing on the pediatric and geriatric age groups. In addition, we will also discuss the biological variable of sex. Characterizing and understanding the mechanistic differences in these distinct HF populations can provide insights that will benefit and personalize therapeutic interventions. Further, we propose that future investigations into the cellular mechanisms involved in the developing and juvenile heart may provide valuable insights for targets that would be beneficial in aging patients.

Acute isoproterenol leads to age-dependent arrhythmogenesis in guinea pigs.

Sudden cardiac death from ventricular arrhythmias is more common in adult patients with with heart failure compared with pediatric patients with heart failure. We identified age-specific differences in arrhythmogenesis using a guinea pig model of acute ß-adrenergic stimulation. Young and adult guinea pigs were exposed to the ß-adrenergic agonist isoproterenol (ISO; 0.7 mg/kg) for 30 min in the absence or presence of flecainide (20 mg/kg), an antiarrhythmic that blocks Na+ and ryanodine channels. Implanted cardiac monitors (Reveal LINQ, Medtronic) were used to monitor heart rhythm. Alterations in phosphorylation and oxidation of ryanodine receptor 2 (RyR2) were measured in left ventricular tissue. There were age-specific differences in arrhythmogenesis and sudden death associated with acute ß-adrenergic stimulation in guinea pigs. Young and adult guinea pigs developed arrhythmias in response to ISO; however, adult animals developed significantly more premature ventricular contractions and experienced higher arrhythmia-related mortality than young guinea pigs treated with ISO. Although there were no significant differences in the phosphorylation of left ventricular RyR2 between young and adult guinea pigs, adult guinea pigs exposed to acute ISO had significantly more oxidation of RyR2. Flecainide treatment significantly improved survival and decreased the number of premature ventricular contractions in young and adult animals in association with lower RyR2 oxidation. Adult guinea pigs had a greater propensity to develop arrhythmias and suffer sudden death than young guinea pigs when acutely exposed to ISO. This was associated with higher oxidation of RyR2. The incidence of sudden death can be rescued with flecainide treatment, which decreases RyR2 oxidation. NEW & NOTEWORTHY Clinically, adult patients with heart failure are more likely to develop arrhythmias and sudden death than pediatric patients with heart failure. In the present study, older guinea pigs also showed a greater propensity to arrhythmias and sudden death than young guinea pigs when acutely exposed to isoproterenol. Although there are well-described age-related cardiac structural changes that predispose patients to arrhythmogenesis, the present data suggest contributions from dynamic changes in cellular signaling also play an important role in arrhythmogenesis.

Differential Response to Heart Failure Medications in Children.

There have been many advances in the treatment of heart failure over the past several years. While these advancements have resulted in improved outcomes in adults with heart failure, these same treatments do not seem to be as efficacious in children with heart failure. Investigations of the failing pediatric heart suggest that there are unique phenotypic, pathologic and molecular differences that could influence how children with heart failure response to adult-based therapies. In this review, several recent studies and the potential implications of their findings on informing the future of the management of pediatric heart failure are discussed.

Fibrosis and Fibrotic Gene Expression in Pediatric and Adult Patients With Idiopathic Dilated Cardiomyopathy.

BACKGROUND: Although fibrosis seems to be prognostic for adverse outcomes in adults with idiopathic dilated cardiomyopathy (IDC), little is known about the prevalence and development of fibrosis in pediatric IDC hearts. We hypothesized that there is less activation of fibrosis at a molecular level in pediatric IDC hearts than in failing adult hearts. METHODS AND RESULTS: Pediatric hearts were analyzed histologically to determine the prevalence of fibrosis. Left ventricular tissue from adult and pediatric IDC hearts and adult and pediatric nonfailing (NF) hearts were subjected to quantitative reverse-transcription polymerase chain reaction to study the expression of important mRNAs that affect fibrosis. We found age-specific differences between IDC and NF hearts in the regulation of noncoding galectin-3, Corin, matrix metalloproteinase (MMP) 2, MMP-9, tissue inhibitor of metalloproteinase (TIMP) 2, and TIMP-3. We also found markers that were similarly altered in both adult and pediatric IDC hearts (interleukin-1 receptor-like 1 receptor, TIMP-1, and TIMP-4). Finally, microRNAs 29a-c were significantly decreased in the pediatric IDC patients. CONCLUSIONS: Pediatric IDC patients demonstrate age-specific differences in the molecular pathways implicated in fibrosis in the adult heart. At the ultrastructural level the unique gene expression pattern appears to limit fibrosis in the failing pediatric heart.

A Laing distal myopathy-associated proline substitution in the ß-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Glycogen synthase kinase-3beta regulates post-myocardial infarction remodeling and stress-induced cardiomyocyte proliferation in vivo.

RATIONALE: Numerous studies have proposed that glycogen synthase kinase (GSK)-3beta is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3beta, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3beta is embryonic-lethal. OBJECTIVE: This study was designed to define the role played by GSK-3beta in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI). METHODS AND RESULTS: We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3beta in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3beta appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3beta does regulate post-MI remodeling because the GSK-3beta knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3beta also led to increased cardiomyocyte proliferation following PO and MI. CONCLUSIONS: Deletion of GSK-3beta protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3beta could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.

Dysfunctional sarcomeric relaxation in the heart.

Since cardiac relaxation is commonly impaired in heart failure caused by many different etiologies, identifying druggable targets is a common goal. While many factors contribute to cardiac relaxation, this review focuses on sarcomeric relaxation and dysfunction. Any alteration in how sarcomeric proteins interact can lead to significant shifts in sarcomeric relaxation that may contribute to diastolic dysfunction. Considering examples of sarcomeric dysfunction that have been reported in 3 different pathologies, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and heart failure with preserved ejection fraction, will provide insights into the role sarcomeric dysfunction plays in impaired cardiac relaxation. This will ultimately improve our understanding of sarcomeric physiology and uncover new therapeutic targets.