Titin Expression Research Articles

Titin hyper-phosphorylation in the peripheral skeletal muscle but not the diaphragm of HFpEF: association with muscle atrophy and dysfunction

Abstract Background Titin hypo-phosphorylation is an important regulatory mechanism for myocardial stiffness in heart failure with preserved ejection fraction (HFpEF). In contrast, a hyper-phosphorylation of titin in the peripheral skeletal muscle (SKM) was reported in mouse models exhibiting muscle atrophy. In former studies it, was shown that SKM atrophy occurs in HFpEF and is associated with muscle dysfunction and a fiber type shift from type I, towards type II fibers. However, these alterations were not seen in the diaphragm muscle (Dia) and a fiber type shift towards type I was documented. So far, no information about the phosphorylation status of titin is available for SKM and the Dia and its association with muscle dysfunction in HFpEF. Purpose To assess titin phosphorylation in SKM and Dia of a HFpEF animal model and to relate it to muscle function and atrophy. Methods To confirm the development of HFpEF echocardiography was performed in female ZSF1-lean (con, n=18) and ZSF1-obese rats (HFpEF, n=18) at the age of 32 weeks. Skeletal muscle function (soleus) was measured in an organ bath setting after the animals were sacrificed. Tissue of the tibialis anterior muscle (TA) and the diaphragm (Dia) was collected and fixed in formalin or snap frozen in liquid nitrogen. To detect titin expression homogenized TA and Dia tissue samples were resolved on a vertical agarose gel. Phosphorylated titin was detected by staining the gel with Pro-Q Diamond, whereas total titin was visualized using Sypro Ruby. Cross sectional area (CSA) of TA and Dia was quantified after Hematoxylin/eosin staining of tissue section. Myosin heavy chain (MHC) ubiquitination was assessed by western blot. Results Assessment of SKM function revealed significantly reduced maximal absolute (-12 %) and maximal specific force (-22 %) in HFpEF animals when compared to controls. In the TA a significantly reduced CSA was evident in HFpEF (1911±103 vs. 1394±103 µm2, p=0.002), whereas in the diaphragm an increased CSA was noted (438±11 vs. 560±18 µm2, p<0.001). In TA of HFpEF animals, the proportion of phosphorylated titin was significantly elevated whereas the total amount of titin was reduced (Fig. 1A, C). In addition, a significantly higher proportion of ubiquitinated MHC was detected (Figure 1E). However in the Dia these alterations were not observed, even a higher amount of total titin was seen (Fig. 1B, D, F). Furthermore, a significant negative correlation between phosphorylated titin and absolute force (r=-0.461, p=0.035, Fig. 2A) as well as specific force (r=-0.693, p=0.0005, Fig. 2B) was evident. Conclusion In contrast to the heart, the TA showed a titin hyper-phosphorylation in HFpEF, going along with a degradation of total titin. This hyper-phosphorylation could be correlated with a loss of force in the SKM. Additionally a higher ubiquitination of MHC was prevalent in the TA, which may be connected to molecular remodeling and fiber type shift towards a more glycolytic type.

Modulation of Titin and Contraction-Regulating Proteins in a Rat Model of Heart Failure with Preserved Ejection Fraction: Limb vs. Diaphragmatic Muscle.

Heart failure with preserved ejection fraction (HFpEF) is characterized by biomechanically dysfunctional cardiomyocytes. Underlying cellular changes include perturbed myocardial titin expression and titin hypophosphorylation leading to titin filament stiffening. Beside these well-studied alterations at the cardiomyocyte level, exercise intolerance is another hallmark of HFpEF caused by molecular alterations in skeletal muscle (SKM). Currently, there is a lack of data regarding titin modulation in the SKM of HFpEF. Therefore, the aim of the present study was to analyze molecular alterations in limb SKM (tibialis anterior (TA)) and in the diaphragm (Dia), as a more central SKM, with a focus on titin, titin phosphorylation, and contraction-regulating proteins. This study was performed with muscle tissue, obtained from 32-week old female ZSF-1 rats, an established a HFpEF rat model. Our results showed a hyperphosphorylation of titin in limb SKM, based on enhanced phosphorylation at the PEVK region, which is known to lead to titin filament stiffening. This hyperphosphorylation could be reversed by high-intensity interval training (HIIT). Additionally, a negative correlation occurring between the phosphorylation state of titin and the muscle force in the limb SKM was evident. For the Dia, no alterations in the phosphorylation state of titin could be detected. Supported by data of previous studies, this suggests an exercise effect of the Dia in HFpEF. Regarding the expression of contraction regulating proteins, significant differences between Dia and limb SKM could be detected, supporting muscle atrophy and dysfunction in limb SKM, but not in the Dia. Altogether, these data suggest a correlation between titin stiffening and the appearance of exercise intolerance in HFpEF, as well as a differential regulation between different SKM groups.

Impact of Nutritional Status of Patients with Head and Neck Squamous Cell Carcinoma on the Expression Profile of Ghrelin, Irisin, and Titin.

The goal of this paper was the evaluation of the changes in the expression profile of irisin, ghrelin, and titin in the carcinoma tissue and in the blood of patients with head and neck squamous cell carcinoma (HNSCC), including determining the profile of their expression in relation to patient nutrition. The study included 56 patients with diagnosed squamous cell carcinoma of HNSCC in the T3 and T4 stages of the disease. Healthy control tissue specimens were collected from an area 10 mm outside the histologically negative margin. In turn, the blood and serum from the control group came from healthy volunteers treated for non-oncologic reasons (n = 70). The molecular analysis allowed us to determine the profile of irisin, ghrelin, and titin methylation, evaluate their expression on the level of mRNA (quantitative Reverse Transcription Polymerase Chain Reaction; qRT-PCR) and protein (Enzyme-Linked Immunosorbent Assay Reaction; ELISA) in the carcinoma tissue and the margin of healthy tissue, as well as in serum of patients in the study and control groups. At the start of our observations, a Body Mass Index (BMI) < 18.5 was noted in 42 of the patients, while six months after the treatment a BMI < 18.5 was noted in 29 patients. We also noted a decrease in the expression of irisin, ghrelin, and titin both on the level of mRNA and protein, as well as a potential regulation of their expression via DNA methylation. There is no convincing evidence that the proteins assayed in the present work are specific with regard to HNSSC.

Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.

Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.

Antifungal mechanisms of the antagonistic bacterium Bacillus mojavensis UTF-33 and its potential as a new biopesticide.

Biological control has gradually become the dominant means of controlling fungal disease over recent years. In this study, an endophytic strain of UTF-33 was isolated from acid mold (Rumex acetosa L.) leaves. Based on 16S rDNA gene sequence comparison, and biochemical and physiological characteristics, this strain was formally identified as Bacillus mojavensis. Bacillus mojavensis UTF-33 was sensitive to most of the antibiotics tested except neomycin. Moreover, the filtrate fermentation solution of Bacillus mojavensis UTF-33 had a significant inhibitory effect on the growth of rice blast and was used in field evaluation tests, which reduced the infestation of rice blast effectively. Rice treated with filtrate fermentation broth exhibited multiple defense mechanisms in response, including the enhanced expression of disease process-related genes and transcription factor genes, and significantly upregulated the gene expression of titin, salicylic acid pathway-related genes, and H2O2 accumulation, in plants; this may directly or indirectly act as an antagonist to pathogenic infestation. Further analysis revealed that the n-butanol crude extract of Bacillus mojavensis UTF-33 could retard or even inhibit conidial germination and prevent the formation of adherent cells both in vitro and in vivo. In addition, the amplification of functional genes for biocontrol using specific primers showed that Bacillus mojavensis UTF-33 expresses genes that can direct the synthesis of bioA, bmyB, fenB, ituD, srfAA and other substances; this information can help us to determine the extraction direction and purification method for inhibitory substances at a later stage. In conclusion, this is the first study to identify Bacillus mojavensis as a potential agent for the control of rice diseases; this strain, and its bioactive substances, have the potential to be developed as biopesticides.

Study of the expression of Cronos titin in TTN-truncation cardiomyopathy and heart development

Cronos is a recently discovered isoform of the giant protein titin driven by an alternative internal promoter. Cronos spans the half-A-band of a sarcomere and binds to myosin filaments, but its role in healthy or diseased hearts is largely unknown. Upregulation of Cronos has been suggested as a possible compensatory mechanism in titin-truncation cardiomyopathy, to help explain why truncations in titin's I-band region are less frequent and often cause a milder disease phenotype than truncations in the A-band region.

Differential Expression of Titin and Obscurin mRNA in Striated Muscles of the Long-Tailed Ground Squirrel Urocitellus undulatus

Differential Expression of Titin and Obscurin mRNA in Striated Muscles of the Long-Tailed Ground Squirrel Urocitellus undulatus

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.

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&amp;lt;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.

Effects of cyclosporine A on proliferation, invasion and migration of HTR-8/SVneo human extravillous trophoblasts

As shown in our previous study, cyclosporine A (CsA) promotes the proliferation, invasion and migration of villous trophoblasts, thus improving embryo implantation. In addition, the incidence of preeclampsia (PE) is decreased in patients with recurrent spontaneous abortion (RSA) and repeated implantation failure (RIF) treated with CsA during the first trimester. Abnormal function of extravillous trophoblasts (EVTs) in early pregnancy is recognized as the pathogenetic mechanism of PE. EVTs share homology and function with pre-villous trophoblasts and villous trophoblasts; thus, we hypothesized that CsA may have the same regulatory effect on EVTs.In this study, we investigated the effects of CsA on HTR-8/SVneo trophoblasts in the extravillous layer and explored the underlying mechanisms. QPCR and Western blot (WB) analyses were performed to detect expression alterations in relevant proliferation and invasion proteins in response to different concentrations of CsA. We used an Affymetrix IVT expression microarray to examine the target genes of CsA in preeclamptic placentas versus normal placentas.Our results showed that certain concentrations of CsA could promote the proliferation, invasion and migration of HTR8/SVneo cells. CsA was also found to promote the expression of titin, MMP9, EGFR, and PRR15. TRAIL may be a target gene for CsA-mediated regulation of EVTs. ConclusionsBy promoting the expression of related proteins and regulating the functions of HTR8/SVneo cells, CsA can promote vascular recasting and placental function, which may affect the pathogenesis of PE.

Moderate Exercise in Spontaneously Hypertensive Rats Is Unable to Activate the Expression of Genes Linked to Mitochondrial Dynamics and Biogenesis in Cardiomyocytes.

Hypertension (HTN) is a public health concern and a major preventable cause of cardiovascular disease (CVD). When uncontrolled, HTN may lead to adverse cardiac remodeling, left ventricular hypertrophy, and ultimately, heart failure. Regular aerobic exercise training exhibits blood pressure protective effects, improves myocardial function, and may reverse pathologic cardiac hypertrophy. These beneficial effects depend at least partially on improved mitochondrial function, decreased oxidative stress, endothelial dysfunction, and apoptotic cell death, which supports the general recommendation of moderate exercise in CVD patients. However, most of these mechanisms have been described on healthy individuals; the effect of moderate exercise on HTN subjects at a cellular level remain largely unknown. We hypothesized that hypertension in adult spontaneously hypertensive rats (SHRs) reduces the mitochondrial response to moderate exercise in the myocardium.Methods: Eight-month-old SHRs and their normotensive control—Wistar-Kyoto rats (WKYR)—were randomly assigned to moderate exercise on a treadmill five times per week with a running speed set at 10 m/min and 15° inclination. The duration of each session was 45 min with a relative intensity of 70–85% of the maximum O2 consumption for a total of 8 weeks. A control group of untrained animals was maintained in their cages with short sessions of 10 min at 10 m/min two times per week to maintain them accustomed to the treadmill. After completing the exercise protocol, we assessed maximum exercise capacity and echocardiographic parameters. Animals were euthanized, and heart and muscle tissue were harvested for protein determinations and gene expression analysis. Measurements were compared using a nonparametric ANOVA (Kruskal-Wallis), with post-hoc Dunn's test.Results: At baseline, SHR presented myocardial remodeling evidenced by left ventricular hypertrophy (interventricular septum 2.08 ± 0.07 vs. 1.62 ± 0.08 mm, p < 0.001), enlarged left atria (0.62 ± 0.1 mm vs. 0.52 ± 0.1, p = 0.04), and impaired diastolic function (E/A ratio 2.43 ± 0.1 vs. 1.56 ± 0.2) when compared to WKYR. Moderate exercise did not induce changes in ventricular remodeling but improved diastolic filling pattern (E/A ratio 2.43 ± 0.1 in untrained SHR vs. 1.89 ± 0.16 trained SHR, p < 0.01). Histological analysis revealed increased myocyte transversal section area, increased Myh7 (myosin heavy chain 7) expression, and collagen fiber accumulation in SHR-control hearts. While the exercise protocol did not modify cardiac size, there was a significant reduction of cardiomyocyte size in the SHR-exercise group. Conversely, titin expression increased only WYK-exercise animals but remained unchanged in the SHR-exercise group. Mitochondrial response to exercise also diverged between SHR and WYKR: while moderate exercise showed an apparent increase in mRNA levels of Ppargc1α, Opa1, Mfn2, Mff, and Drp1 in WYKR, mitochondrial dynamics proteins remained unchanged in response to exercise in SHR. This finding was further confirmed by decreased levels of MFN2 and OPA1 in SHR at baseline and increased OPA1 processing in response to exercise in heart. In summary, aerobic exercise improves diastolic parameters in SHR but fails to activate the cardiomyocyte mitochondrial adaptive response observed in healthy individuals. This finding may explain the discrepancies on the effect of exercise in clinical settings and evidence of the need to further refine our understanding of the molecular response to physical activity in HTN subjects.

Single-cell RNA-sequencing of leprosy skin lesions reveals a titin (TTN) expressing T cell subset associated with the progressive disease

Abstract The immune response to intracellular bacteria is critical for host defense against the invading pathogen. This interaction can be studied in the human disease leprosy caused by the intracellular bacterium Mycobacterium leprae affecting primarily the skin compartments. Patients with tuberculoid leprosy (T-lep) express a Th1 cytokine pattern in skin lesions and develop a protective immunity that eliminates the infection, whereas those with lepromatous leprosy (L-lep) express Th2 cytokines in lesions and sustain a progressive infection. In the reversal reaction (RR) state, patients upgrade, either spontaneously or during chemotherapy, from the L-lep to the T-lep pole. Herein, we used unbiased single-cell RNA-sequencing to explore the breadth of potential immune cell states in leprosy. Unexpectedly, we identified a lymphocyte cluster marked by expression of titin (TTN), which is commonly found in the sarcomere of muscle cells. TTN+ T cells were predominantly enriched in L-lep lesions and RNA-FISH showed a higher number of TTN mRNA dots on CD3+ T cells from L-lep in comparison with RR patients. TTN gene expression decreased in the skin lesion cells of L-lep patients undergoing RR episodes associated with the onset of therapy. Protein expression was confirmed by analysis of T cell clones isolated from leprosy lesions and blood-derived CD3+ T cells from healthy donors. Indeed, immunohistochemical staining of lesions and healthy tonsil showed that TTN was present in the CD3+ T cell zones. Canonical pathways analysis revealed that TTN might be involved with T cell inhibitory RhoA signaling pathway. The role of TTN on T cells remains unknown, however, it could represent a novel mechanism in the skin immune response against cutaneous pathogens.

Effect of L-Arginine on Titin Expression in Rat Soleus Muscle After Hindlimb Unloading.

Nitric oxide (NO), produced by NO-synthases via L-arginine oxidation, is an essential trigger for signaling processes involved in structural and metabolic changes in muscle fibers. Recently, it was shown that L-arginine administration prevented the decrease in levels of the muscle cytoskeletal proteins, desmin and dystrophin, in rat soleus muscle after 14 days of hindlimb unloading. Therefore, in this study, we investigated the effect of L-arginine administration on the degree of atrophy changes in the rat soleus muscles under unloading conditions, and on the content, gene expression, and phosphorylation level of titin, the giant protein of striated muscles, able to form a third type of myofilaments—elastic filaments. A 7-day gravitational unloading [hindlimb suspension (HS) group] resulted in a decrease in the soleus weight:body weight ratio (by 31.8%, p < 0.05), indicating muscle atrophy development. The content of intact titin (T1) decreased (by 22.4%, p < 0.05) and the content of proteolytic fragments of titin (T2) increased (by 66.7%, p < 0.05) in the soleus muscle of HS rats, compared to control rats. The titin gene expression and phosphorylation level of titin between these two groups were not significantly different. L-Arginine administration under 7-day gravitational unloading decreased the degree of atrophy changes and also prevented the decrease in levels of T1 in the soleus muscle as compared to HS group. Furthermore, L-arginine administration under unloading resulted in increased titin mRNA level (by 76%, p < 0.05) and decreased phosphorylation level of T2 (by 28%, p < 0.05), compared to those in the HS group. These results suggest that administration of L-arginine, the NO precursor, under unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle.

PO-078 Effects of aerobic and resistance training on cardiac diastolic dysfunction in type 2 diabetic rats

Objective To compare the effects of two types of physical training on cardiac diastolic dysfunction in type 2 diabetes mellitus and investigate the role of passive tension regulators—titin and collagen.&#x0D; Methods Sprague-Dawley (SD) male rats were divided into two groups: control and diabetes. Each group was subdivided into three subgroups: sedentary rats, rats submitted to aerobic trained and rats submitted to resistance training. Diabetic rats were induced by high-fat diet combined with low-dose injections of streptozocin (STZ). Aerobic trained rats ran on a treadmill at 21m/min for 60 min per day, while resistance trained rats climbed a ladder with incremental loads. The train period lasted for 8 weeks.&#x0D; Results All diabetic rats had increased fasting blood glucose (FBG) and left ventricular end diastolic pressure (LVEDP), a lower down stroke of the pressure curve (dp/dt min), longer exponential time constants of relaxation (Tau) in relation to control rats, while the protein expression of titin was decreased significantly, and the expression of collagen I, TGFβ1 were increased slightly, the ratio of type I and III collagen was raised in diabetic rats. Both types of training promoted a decrease in FBG and HOMA-IR in diabetic rats. Aerobic exercise trained diabetic rats had significant higher –dp/dtmin, and shorter Tau, but -dp/dtmin and Tau in resistance exercise trained diabetic rats had no significant been improved, even more aggravation. Moreover, aerobic training increased the protein expression of titin and HSP27, and the amount of co-localization of titin and HSP27 elevated, the protein expression of collagen I, TGFβ1 were decreased, and the ratio of type I and type III got close to normal. Resistance training further decreased the expression of titin, collagen I, and TGFβ1, collagen volume fraction (CVF) was increased significantly, and the ratio of type I and III collagen was disturbed, but the co-localization of titin and HSP27 elevated slightly.&#x0D; Conclusions Aerobic training ameliorates cardiac diastolic dysfunction in diabetes and this improvement may be related to increase titin repaired by HSP27, while resistance training aggravates the cardiac diastolic dysfunction in early diabetes mellitus and it could be caused by worsening myocardial interstitial fibrosis.

A18878 Circular RNA expression is reset by brief antihypertensive treatment in spontaneously hypertensive rats (SHR)

Objectives: The ‘legacy effect’ of persistently lower blood pressure in SHR is observed after short-term treatment with angiotensin converting enzyme inhibitor (ACEi) or angiotensin II receptor blocker (ARB) antihypertensive drugs and is associated with changes in renal function. This presumably involves resetting of the genetic program leading to hypertension in SHR. Circular RNAs (circRNA) are novel non-coding RNAs that regulate mRNA expression post-transcriptionally and might be relevant to the legacy effect. Our aim was to study circRNA expression in the SHR after antihypertensive treatment. Methods: We treated male SHR with an ACEi (perindopril), an angiotensin II receptor blocker (losartan) or a vasodilator (hydralazine) from 10–14 weeks of age. Renal cortex and cardiac left ventricular tissues were collected at 20-weeks of age (n = 3–5 per group). We measured the expression of heart related circular RNA (Hrcr), circular titin (cTtn) and circular foxhead box O3 (cFoxo3) using quantitative real-time PCR. These circRNAs have been implicated in cardiac hypertrophy and heart failure in mice. Statistical analysis was performed using one-way ANOVA and significance was set as P < 0.05. Results: Hrcr and cFoxo3 were downregulated in renal cortex at 20-weeks when compared with vehicle after all antihypertensive treatments (P < 0.01 for all). However, renal cortical cTtn expression was upregulated only after treatment with ACEi (P < 0.05) or ARB (P < 0.05). Differences in circRNA expression were not observed in heart tissues. Conclusion: Early antihypertensive treatment in SHR resets circRNA expression in renal cortices of SHR but not in the heart. This indicates tissue-specific regulation of circRNA expression might be important in reprogramming genetic hypertension by early renin-angiotensin system blocking drugs in SHR.

Diabetes-Induced Cardiomyocyte Passive Stiffening Is Caused by Impaired Insulin-Dependent Titin Modification and Can Be Modulated by Neuregulin-1.

Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E-deficient mice with streptozotocin-induced diabetes mellitus (n=12-22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5-6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKCα (protein kinase Cα) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKCα activity. In apolipoprotein E-deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKCα activity, which reversed the diabetes mellitus-associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. Mechanistically, we found that impaired cGMP-PKG signaling and elevated PKCα activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKCα, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.

Assessment of Cardiac Function of Rbm 20−/− Rats Using Pressure‐Volume Loop Analysis After 28 Days of Angiotensin II Treatment

RNA binding motif 20 (Rbm20), a muscle specific splicing factor for the sarcomeric protein titin, affects diastolic and systolic properties of the heart per the Frank‐Starling mechanism. Failure to alter titin expression from the predominant fetal isoform N2BA to the shorter, adult N2B isoform in Rbm20−/− (KO) rats, results in impaired cardiomyocyte relaxation, increased compliance of the ventricular walls, and dilated cardiomyopathy. These pathologies are similar to those seen in humans with this mutation, which often results in early, sudden death in both species. Angiotensin II (AII) acutely causes vasoconstriction, and chronically results in cardiac hypertrophy, characterized by an increase in cardiac wall stiffness. We hypothesized that 28 days of treatment with AII would increase the stiffness of the myocardium, resulting in improved cardiac function in KO rats. KO rats and age‐matched wild type (WT) controls were fitted with subdermal osmotic pumps, delivering AII continuously (0.25 μL·hr −1) for 28 days. After AII exposure, cardiac function was assessed in vivo using pressure‐volume loop analysis. Under isoflurane anesthesia, loops were recorded under baseline, brief inferior vena cava occlusion, and hypertonic saline infusion (20–40 μL) conditions. The rat was then heparinized, and blood collected for cuvette calibration. No group differences (p&lt;0.05) were found in cardiac output (23.3±0.8 KO; 26.9±2.5 mL·min−1 WT), heart rate (295±21 KO; 341±16 b·min−1 WT), and stroke volume (80.8±9.1 KO; 79.5±7.6 μL WT). However, significant reductions were found in contractility (preload recruitable stroke work; 41.6±10.8 KO; 86.9±8.2 mmHg WT), stroke work (5.13±0.73; 8.13±0.86 mmHg·min WT), and ejection fraction (49.8±4.2 KO; 74.7±7.4 % WT) in KO rats. KO rats were able to maintain stroke volume, despite impaired contractility, due to a significant increase in preload (169±16 KO; 113±7 μL WT) and decrease in afterload (84.4±8.7 KO; 113±9 mmHg WT). We conclude that KO rats failed to improve contractility with 28 days of AII treatment, but maintained normal cardiac output through compensatory changes in preload and afterload.Support or Funding InformationThis project is supported in part by a grant from the National Institute of General Medical Sciences (2P20GM103432) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Skeletal Muscle Sarcomere Length through Titin Changes in iMSBmal1 Mice

Prior research shows that sarcomere length correlates with isometric force production, but the specific structural elements regulating resting sarcomere length are not well understood. Based on the assumption that sarcomeres operate similarly throughout a muscle fiber, heterogeneity in sarcomere lengths should decrease maximum active tension production. One structural protein that has been proposed to maintain sarcomere length is titin. Our lab developed an inducible skeletal muscle specific Bmal1 knockout mouse, which exhibits muscle weakness and a shift to longer titin isoform. My project takes a histological approach to study phenotypic changes in sarcomere length and A‐band centrality in the iMSBmal1−/− skeletal muscle. I hypothesized that the iMSBmal1−/− mice would exhibit an increased variance in sarcomere length, as well as a change in the A‐band centrality index (i.e. the distance of peak fluorescence of the myosin filament from the center of the sarcomere). After collecting tibilalis anterior (TA) muscle from these mice at 5 weeks after recombination, the muscles were frozen in OCT compound using liquid nitrogen cooled isopentane. These samples (n=3 animals/group) were then longitudinally cryosectioned at 6 microns. The samples were stained using primary antibodies that target α‐actinin and myosin heavy chain followed by fluorescent secondary antibodies. α‐actinin labels the Z‐lines, allowing us to measure sarcomere lengths in the longitudinal sections, while myosin heavy chain allows us to visualize the A‐band. I measured the peak fluorescence of both fluorescent secondary antibodies and measured where the myosin heavy chain peak was in relation to the Z‐line on either side to determine an A‐band centering index. The data was compiled by sample (~450 sarcomeres measured per sample) and then averaged both by sample and then by comparing iMSBmal1+/+ to iMSBmal1−/−. Results of iMSBmal1+/+ to iMSBmal1−/− showed a trend toward a longer sarcomere length from 2.64 μm to 2.82 μm (not significant, p=0.1), but the variance did not change. Since the variance of sarcomere lengths did not change, my hypothesis that altered titin expression correlates to more variable sarcomere length was not supported; however, longer titin could lead to the observed increased sarcomere length and my sample number is currently small (n=3/group). The mean and variance of the A‐band centrality index between these groups were not found to be statistically significant (p = 0.7769 and 0.3672, respectively). The lack of change in the A‐band centrality index does not support my hypothesis that a shift would occur. However, there were some technical issues with the antibody that I am repeating and further studying. Further investigations will include increasing my sample size with a focus on sarcomere length. I will also be using site specific titin antibodies to look at the inclusion of longer titin isoforms through the sarcomeres in muscle. Understanding the relationship among titin isoforms, sarcomere length and structure and muscle force will be important for understanding causes of muscle weakness.Support or Funding InformationThis work was made possible by the University of Florida and the funding of the American Physiological Society Undergraduate Summer Research Fellowship Program. Special thanks to Lance A. Riley and Karyn A. Esser for their support and guidance through this process. Thanks also to David Hammers and Cora Coker for their help with microscopy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis.

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.

CsA Promotes XIST Expression to Regulate Human Trophoblast Cells Proliferation and Invasion Through miR-144/Titin Axis.

In our previous study, we revealed that Cyclosporin A (CsA) could inhibit miR-144 expression to regulate proliferation and invasion of human trophoblast (HT) cells through miR-144 targeting titin. This partially demonstrated the mechanism by which CsA promotes titin expression to increase the vitality of HT cells. However, the mechanism by which CsA inhibits miR-144 expression remains to be investigated. Recently, the interaction between lncRNA and miRNA has been frequently reported to play major role in several biological processes. In the present study, online tools were used to figure out that X-inactive specific transcript (XIST) could interact with miR-144. XIST and miR-144 reciprocally inhibited each other in HT cells; as exhibited by luciferase reporter gene assays, miR-144 bind to XIST by direct targeting. XIST suppressed miR-144 expression to promote titin expression. As exhibited by the Spearman's correlation analysis, in CsA treated HT cells, miR-144 was inversely correlated with titin and XIST, respectively; XIST was positively correlated with titin. Moreover, CsA could promote the proliferation and invasion of HT cells through XIST and the downstream MAPK and MMPs pathway. Taken together, these findings will shed light to the role and mechanism of CsA/XIST/miR-144/titin in regulating HT cells proliferation and invasion. XIST may serve as a potential therapeutic target in HT in the future. J. Cell. Biochem. 118: 2208-2218, 2017. © 2017 Wiley Periodicals, Inc.