Adult Cardiac Muscle Research Articles

Drosophila as a Model to Understand Second Heart Field Development.

The genetic model system Drosophila has contributed fundamentally to our understanding of mammalian heart specification, development, and congenital heart disease. The relatively simple Drosophila heart is a linear muscular tube that is specified and develops in the embryo and persists throughout the life of the animal. It functions at all stages to circulate hemolymph within the open circulatory system of the body. During Drosophila metamorphosis, the cardiac tube is remodeled, and a new layer of muscle fibers spreads over the ventral surface of the heart to form the ventral longitudinal muscles. The formation of these fibers depends critically upon genes known to be necessary for mammalian second heart field (SHF) formation. Here, we review the prior contributions of the Drosophila system to the understanding of heart development and disease, discuss the importance of the SHF to mammalian heart development and disease, and then discuss how the ventral longitudinal adult cardiac muscles can serve as a novel model for understanding SHF development and disease.

Switching ubiquitous and muscle-specific isoforms of mitochondrial respiratory complex IV in skeletal muscle fine-tunes complex IV activity.

Respiratory complex IV (CIV, cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain. Some CIV subunits have two or more isoforms, which are ubiquitously expressed or are expressed in specific tissues like the lung, muscle, and testis. Among the tissue-specific CIV isoforms, the muscle-specific isoforms are expressed in adult cardiac and skeletal muscles. To date, the physiological and biochemical association between the muscle-specific CIV isoforms and aerobic respiration in muscles remains unclear. In this study, we profiled the CIV organization and expression pattern of muscle-specific CIV isoforms in different mouse muscle tissues. We found extensive CIV-containing supramolecular organization in murine musculature at advanced developmental stages, while a switch in the expression from ubiquitous to muscle-specific isoforms of CIV was also detected. Such a switch was confirmed during the in vitro differentiation of mouse C2C12 myoblasts. Unexpectedly, a CIV expression decrease was observed during C2C12 differentiation, which was probably due to a small increase in the expression of muscle-specific isoforms coupled with a dramatic decrease in the ubiquitous isoforms. We also found that the enzymatic activity of CIV containing the muscle-specific isoform COX6A2 was higher than that with COX6A1 in engineered HEK293T cells. Overall, our results indicate that switching the expression from ubiquitous to muscle-specific CIV isoforms is indispensable for optimized oxidative phosphorylation in mature skeletal muscles. We also note that the in vitro C2C12 differentiation model is not suitable for the study of muscular aerobic respiration due to insufficient expression of muscle-specific CIV isoforms.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Dynamic nuclear-sarcomeric coupling in the adult cardiac muscle

Dynamic nuclear-sarcomeric coupling in the adult cardiac muscle

Lipid-independent activation of a muscle-specific PKCα splicing variant.

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.NEW & NOTEWORTHY PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of Prkca gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete’s heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin’s role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete’s heart. The athlete’s heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete’s heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Tbx5 navigates through the labyrinth of adult cardiac regeneration

Abstract Background Heart failure is the major cause of death and morbidity in industrialized countries with an estimated 23 million people affected per year, representing 30% of all global deaths. Injury to the adult mammalian cardiac muscle, often leads to a heart attack due to irreversible loss of a large number of cardiomyocytes (CM) and other cardiac interstitial cells, creating an unmet need for identifying a cardiac progenitor cell (CPC) population for cardiac replenishment. In contrast, amphibians and neonatal rodents possess the ability to regenerate their heart upon injury. It has been suggested recently that idle cardiac regenerative mechanisms may be present in adult mammals, inhibited by exogenous cues, or lack of. Murine and human CPCs can be isolated through the expression of Pdgfra, Kdr, and our novel surface marker, Gfra2. In addition, the expression of the embryonic transcription factor TBX5, is paramount for differentiation towards a cardiomyocyte fate. Therefore, Tbx5-expressing CPCs could be an effective target for proof-of-concept studies in the heart repair field, inclined to pharmacological modulation in patients with ischemic heart disease. Purpose To characterise an adult Tbx5-expressing CPC population in the injured heart. Using a developmental approach to two adult heart injury murine models, we show that Tbx5-expressing CPC exist in the injured adult mammalian heart, with a molecular signature that strongly correlates with that of embryonic and neonatal CPCs. Methods A well-defined tamoxifen-induced Tbx5-Cre; Rosa26R-eYFP/eYFP transgenic mouse model was employed, where myocardial infarction (MI) was induced through reperfusion/ischemia or chemical injury. Cardiac cells expressing YFP+ cells were collected from the adult injured hearts five to seven days post-injury. Flow-cytometric and single-cell mRNA-seq analysis was performed in order to collect and compare those YFP+ cells to control adult uninjured cardiac interstitial cells, and early neonatal-derived CPCs. Results Immunohistochemical analysis indicated that YFP+ interstitial cells were mostly present in the injury site, but also in distal cardiac areas. Flow cytometric analysis of live cells pinpoint these YFP+ cells are part of a CPC-like population. Single-cell mRNA transcriptomic analysis revealed that YFP+ cells resemble early postnatal CPC. Yet, YFP+ cells never expressed CM markers in vivo, but they did differentiate into CM-like cells, in vitro. Conclusions Upon MI, the adult heart possess an interstitial cell population that transiently re-activates the pioneer cardiac embryonic transcription factor Tbx5. The Tbx5-expressing cell population transcriptomically resembles that of CPC, which could promote CM regeneration upon neonatal injury. We show that Tbx5 lies in the centre of a novel adult CPC population. The adult heart microenvironment may hinder mammalian CM regeneration through regulation of the Tbx5-induced cardiac gene program. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Foundation for Research & Innovation

The stress responsive gene ankrd1a is dynamically regulated during skeletal muscle development and upregulated following cardiac injury in border zone cardiomyocytes in adult zebrafish.

Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.

Postnatal development of the mitochondrial reticulum in the cardiac muscles of mice

Adult cardiac muscles are characterized by well-developed mitochondrial networks, which supports high energy demand in the heart. However, it is obscure how these structures are formed during postnatal development. Using focused ion beam scanning electron microscopy (FIB-SEM), we collected high-resolution image stacks of cardiac muscles of mice at postnatal (P) day 1, 7, 14, and 42, respectively (N=2-3 per group). Then, we conducted a semi-automated machine running to extract 3D mitochondrial formations and analyzed its morphological characteristics across postnatal days. After forming more stretched structures by P7, it was shown that cardiac muscles further develop spherical shaped-mitochondria and that they are overall arranged in the fiber-parallel direction. Although total mitochondrial volumes tend to increase across P1-P42, individual mitochondrial volumes were relatively smaller during early postnatal days (P1-P14) than in the matured muscles. Notably, we observed that individual mitochondrial surface area to volume ratios as well as aspect ratios are significantly downregulated during the entire developmental period by ~34% and ~31%, respectively. Furthermore, it was also shown that individual mitochondrial lengths are ~31% decreased across the postnatal days. In conclusion, these preliminary results provide dynamic transitions of individual mitochondrial structures during postnatal cardiac muscle development. Nevertheless, further analyses will be needed to study intermitochondrial interactions, as well as mitochondrial interactions with other subcellular components (e.g., lipid droplets), by which we can better understand functional aspects of mitochondria in developing cardiac muscles.

Evaluation of cardiac titin expression after long-term exercise in a rat model

Abstract Introduction The main role of the giant elastic protein titin is to provide passive stiffness to striated (i.e. cardiac and skeletal) muscles. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B isoforms. Titin reduces passive stiffness in cardiac muscle by increased expression of the more compliant N2BA isoform (elevated N2BA:N2B ratio). Moreover, decreased passive stiffness is associated with increased exercise tolerance. Long-term exercise induces physiological adaptation of the heart, termed athlete's heart. Currently, there is limited data of titin's role in the athlete's heart. Aims Our aim is to evaluate exercise-induced morphological and functional changes of the heart. Furthermore, to determine the N2BA:N2B ratio in the rat model of athlete's heart. Methods Rats were divided into exercised (n=12) and control (n=12) groups. Athlete's heart was induced by a 12-week-long swim training (200min/day). The control group swam 5 min/day. Following the training period cardiac changes were assessed by echocardiography. In vivo cardiac function was examined by left ventricular (LV) pressure-volume (P-V) analysis. Titin isoform expressions were detected by sodium-dodecyl-sulfate (SDS)-agarose gel electrophoresis. Results Echocardiography and post-mortem measured cardiomyocyte diameters confirmed athlete's heart and LV hypertrophy in exercised rats. P-V analysis showed improved contractility, active relaxation and mechanoenergetics in the exercised group. The N2BA:N2B titin ratio was significantly increased in exercised rats compared to controls (0.28 vs. 0.21, p<0.05). Conclusions Our results confirm the morphological and functional changes of the athlete's heart. The increased ratio of N2BA:N2B titin corresponds to a more compliant heart in the exercised rats. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Research, Development and Innovation Office of Hungary (K 120277) and the ÚNKP-19-3-I New National Excellence Program of The Ministry For Innovation and Technology.

Differential effect of high fat diet (HFD) on the cardiac muscle of adult and aged female mice and the possible protective role of artichoke treatment: Histomorphometric and ultrastructural study

Background: Coexistence of hyperlipidemia and aging is a highly serious condition that massively deteriorates cardiacfunction. Artichoke is a well-known antioxidant commonly used in long-lived countries diet. Recent researches showed itshypolipidemic and antiaging effects.Objective: This work aimed to investigate the histopathological effects of high fat diet (HFD) on adult mice cardiac muscle, toaddress and compare the extent of myocardial damage in aged mice and to assess beneficial effects of artichoke in mitigatingthese effects.Materials and Methods: A total of 42 adult (Group I) and aged (Group II) female mice (3 and 26 months old respectively)were divided into six equal subgroups (7 per each); Groups Ia and IIa: adult and aged control, Groups Ib and IIb: adult andaged HFD groups were fed diet enriched with 1.5% cholesterol and 10% lard oil for 6 weeks. Groups Ic and IIc: adult andaged artichoke treated group, were received HFD for 6 weeks as HFD groups concomitant with artichoke extract at a dose of1.5 gm/kg once daily via gastric tube.Results: Adult HFD laden cardiac myocytes showed loss of cross striations, inflammatory cell infiltrate, increase collagencontent, myofiber focal degeneration,disruption of myofibrils, sarcomeres and T tubule system, lipid vacuolation, mitochondrialdamage and nuclear pyknosis. These effects were the worst in aged HFD fed animals. Artichoke markedly improved thesechanges in both adult and aged mice.Conclusion: combination of HFD and aging caused more extensive insults on cardiac myocytes than each factor alone.Artichoke is highly efficient in ameliorating these insults.

Developmental increase in β-MHC enhances sarcomere length-dependent activation in the myocardium.

Shifts in myosin heavy chain (MHC) isoforms in cardiac myocytes have been shown to alter cardiac muscle function not only in healthy developing hearts but also in diseased hearts. In guinea pig hearts, there is a large age-dependent shift in MHC isoforms from 80% α-MHC/20% β-MHC at 3 wk to 14% α-MHC/86% β-MHC at 11 wk. Because kinetic differences in α- and β-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and β-MHC expression in guinea pig cardiac muscle impact sarcomere length (SL)-dependent contractile function. We therefore measure steady state and dynamic contractile parameters in detergent-skinned cardiac muscle preparations isolated from the left ventricles of young (3 wk old) or adult (11 wk old) guinea pigs at two different SLs: short (1.9 µm) and long (2.3 µm). Our data show that SL-dependent effects on contractile parameters are augmented in adult guinea pig cardiac muscle preparations. Notably, the SL-mediated increase in myofilament Ca2+ sensitivity (ΔpCa50) is twofold greater in adult guinea pig muscle preparations (ΔpCa50 being 0.11 units in adult preparations but only 0.05 units in young preparations). Furthermore, adult guinea pig cardiac muscle preparations display greater SL-dependent changes than young muscle preparations in (1) the magnitude of length-mediated increase in the recruitment of new force-bearing XBs, (2) XB detachment rate, (3) XB strain-mediated effects on other force-bearing XBs, and (4) the rate constant of force redevelopment. Our findings suggest that increased β-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.

Apoptotic cell death in heart failure associated with diabetes

Diabetes is a serious public health problem. Improvements in the treatment of noncardiac complications from diabetes have resulted in heart disease becoming a leading cause of death in diabetic patients. Several cardiovascular pathological consequences of diabetes such as hypertension affect the heart to varying degrees. Furthermore, hyperglycemia as a sole risk factor may directly cause cardiovascular damage and lead to a complication such as diabetic cardiomyopathy. Alterations in myocardial structure and function occur during the late stage of diabetes. These chronic alterations are believed to result from acute cardiac responses to suddenly increased glucose levels at the early stage of diabetes. In failing hearts, cardiomyocytes degenerate and interstitial fibrosis, which indicates cardiomyocyte loss, becomes more prominent in the myocardium. Various mechanisms may establish an association of diabetes mellitus and heart failure as follows: first, comorbidities such as hypertension may play a role, diabetes also accelerates the development of coronary atherosclerosis, and experimental, clinical studies support the existence of microangiopathy, metabolic factors, or myocardial fibrosis influencing diabetic cardiomyopathy. Apoptosis of adult cardiac muscle cells can have lasting adverse consequences on overall cardiac performance. It is possible that autophagic degeneration may also be one of the mechanisms of myocardial cell death.

The Prophylactic Role of Vitamin E on the Alternations of Histology and Intermediate Filaments of Aged Rabbit cardiomyocytes

The present study is designed to investigate the accumulative effect of aging on the histological and immunohistochemical (IHC) changes of rabbit's cardiac muscle and the protective role of vitamin E on senescent animals. The study was carried out on male New Zeland rabbits; the animals were divided into three groups; group I served as adult rabbits (6 months age, weighing 4 ± 0.5 kg), group II: aged rabbits (24 month age, weighing 7.5 ± 0.5 kg) and group III aged animals supplemented daily with the curative dose of vitamin E orally at 10 mg/kg b.w/day for 60 days. Histological study of the adult cardiac muscle revealed normal branched cardiomyocytes and normal striations with normal oval centrally located nuclei. The aged rabbits showed changes in the cardiac muscle structure that represented as disorganization of the cardiomyocytes with pyknotic nuclei, necrotic areas in the degenerated muscle fibers, congestion and dilatation of blood vessels. By IHC study, the cardiac muscle of adult rabbits expressed moderate normal desmin immunostain in the intercalated discs and Z-lines of myofibres, and moderate normal vimentin immunostain in the connective tissue and periphery to blood vessels in endomysium between cardiac myocytes. The aged rabbit's cardiac muscle expressed an intense immunopositive reaction to both desmin and vimentin. Administrative curative dose of vitamin E as anti-aging medication, antagonize the deleterious effects of aging and improved the architecture of cardiac muscle that have been confirmed through the histological and IHC investigations in the present study.

Gestational protein restriction: study of the probable effects on cardiac muscle structure and function in adult rats.

Intrauterine growth restriction (IUGR) has been linked to heart disease in adulthood. This study aimed to examine the effect of gestational protein restriction during fetal and early postnatal life on the cardiac muscle structure and function in adult offspring. Pregnant female rats were randomly divided into two dietary groups: normal-protein diet (NP) and low-protein diet (LP). Fifteen male offspring from each group were included in the study. Offspring body weights were recorded at birth and monthly from weaning until 24 weeks of age while systolic blood pressure was measured weekly. At the end of the experiment, hearts were weighed and processed for light and electron microscopy and immunohistochemical study. Immunohistochemical staining for localization of inducible nitric oxide synthase (iNOS) and connexin 43 proteins was performed. The gestational protein restriction induced deleterious effects on adult offspring including decreased birth weight, heart weight, and heart rate, and increased systolic blood pressure. Histologically, the number of cardiomyocytes decreased and cardiac fibrosis increased. Signs of degeneration at both structural and ultra-structural levels of cardiomyocytes were also seen. The iNOS was up regulated in LP offspring which was a promoter for apoptosis, while connexin 43 was down regulated which would affect heart conductivity and contractility. Our results demonstrate that adult offspring body weight and cardiac muscle structure and function can be programmed by maternal gestational nutrition. These adverse outcomes suggest the criticality of dietary behavior during pregnancy on long-term offspring cardiac health.

Study of bovine gene: the temporal-spatial expression patterns, polymorphism and association analysis with meat production traits.

The gene () encodes a transcription factor belonging to the MEF2 family that plays an important role in myogenesis by transcriptional regulation of genes involved in skeletal muscle growth and development. Despite the established importance of the factors in the muscular growth and development, the temporal-spatial expression and biological function of have not been reported in cattle. The aim of this study was to analyze the level of expression in the developing longissimus dorsi muscle (LM) of 4 cattle breeds (Polish Holstein-Friesian [HF], Limousine [LIM], Hereford [HER], Polish Red [PR]), differing in terms of meat production and utility type, at 6, 9, and 12 mo of age. The genetic polymorphism and expression patterns in 6 tissues (heart, spleen, liver, semitendinosus muscle [ST], gluteus medius muscle [GM], and LM) were also investigated. The results showed that mRNA was expressed at a high level in adult skeletal and cardiac muscles. Moreover, expression was markedly greater in the GM than in the LM ( 0.05) and ST ( 0.01). An age-dependent and breed-specific comparison of mRNA level in skeletal muscle of HF, LIM, HER, and PR bulls showed that age was significant differentiating factor of transcript/protein abundance in the LM of HER and LIM ( 0.001) compared to HF and PR, for which the differences in mRNA level were not significant ( > 0.05). Regarding the breed effect on the expression, significantly greater mRNA/protein level was noticed in the LM of 9 and 12 mo-old HER than of LIM ( 0.01), HF ( 0.001), and PR ( 0.001). Four novel SNP, namely, (promoter), (exon 7), (exon 8), and (3'UTR), were identified. We found that 3'UTR variant, situated within the seed region of the miR-5187-3p and miR-6931-5p binding sites, was associated with the level of mRNA/protein in LM of 12-mo-old HF bulls. In addition, we observed a significant association between some carcass quality traits, including meat and carcass fatness quality traits, and various 3'UTR genotypes in the investigated population of HF cattle. Our finding provides new evidence of the significant role in the postnatal muscle growth and development in cattle, and indicates that can be a promising molecular marker for carcass quality-related traits in adult cattle.

MTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

Silk-extracellular matrix sponges for striated muscle tissue engineering: Regeneration after injury and disease

Event Abstract Back to Event Silk-extracellular matrix sponges for striated muscle tissue engineering: Regeneration after injury and disease Whitney Stoppel1, Kelly E. Sullivan1, David L. Kaplan1 and Lauren D. Black1, 2 1 Tufts University, Biomedical Engineering, United States 2 Sackler School of Graduate Biomedical Sciences at Tufts University, Cell, Molecular, and Developmental Biology, United States Introduction: Repair and regeneration of critically sized defects or injuries to striated muscle tissue, such as cardiac and skeletal muscles, necessitates the use of a complex biomaterial capable of providing structural support that mimics native tissue mechanics and architecture, while promoting host cell integration, vascularization, and remodeling. Silk fibroin protein sponges containing decellularized extracellular matrix (ECM) make an excellent candidate material for the support and repair of injured tissue due to the tunability of the silk scaffold properties, the ease of incorporation of ECM components[1], and demonstrated efficacy in vitro[3],[4]. Materials and Methods: Silk fibroin protein was extracted from B. mori cocoons[2]. ECM was isolated from either adult or fetal porcine cardiac muscle. Anisotropic sponges were formed via directional freezing, followed by lyophilization and water annealing (Fig. 1). Primary and stem cell-derived cardiac progenitors were used to evaluate scaffold formulations in vitro via Western blot analysis and histology. Optimized scaffolds were implanted subcutaneously in Sprague Dawley rats to evaluate cell infiltration and vascularization[1]. In addition, scaffolds were implanted at the site of injury (left ventricle following permanent ligation of the left anterior descending coronary artery to induce myocardial infarction (MI)). Ejection fraction and left ventricle diameters were used to monitor animal health and assess functional outcomes over 8 weeks (echocardiography). Pressure-volume loop analyses, echocardiography, histology, and gene expression of cells in the infarct region and boarder zones were used to determine the degree of repair, cell infiltration, vascularization, and immune response at the termination of the study. All silk-ECM sponges were compared to a silk only control as well as a sham (no repair) condition. Results and Discussion: Addition of ECM components improved cell infiltration in silk sponges implanted subcutaneously[1]. Silk-ECM composite patches also showed improved function of cardiac progenitors in vitro. In a pilot study, trends in ejection fraction and fractional area shortening calculated from echocardiography data suggested that silk-ECM patches were able to restore function similar to pre-injury values after 6 and 8 weeks (p < 0.15) (Fig. 2). Ongoing experiments are evaluating silk-ECM sponges loaded with soluble ECM for their ability to improve cardiac function following myocardial infarction (1 week post-MI implant). Conclusion: Silk fibroin-ECM sponges improve host cell infiltration and remodeling, promote angiogenesis, and improve functional measures in rodent models of cardiac injury. Future work aims to develop materials for the eventual use in the repair of congenital heart defects in pediatric patients. This work was funded in part by the National Institutes of Health P41 program (EB002520).; WLS would like to acknowledge funding from the National Institutes of Health Institutional Research and Academic Career Development Awards program at Tufts University (K12GM074869, Training in Education and Critical Research Skills (TEACRS)).

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes.

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure.

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.