An Optimized Protocol for Human Direct Cardiac Reprogramming.

Direct cardiac reprogramming, the conversion of fibroblasts into cardiomyocyte-like cells (iCMs), is an attractive approach to heal the injured heart. Here we present a new approach to human cardiac reprogramming that utilizes a polycistronic three-factor reprogramming cocktail and one microRNA. Our protocol produces cardiac Troponin T positive human iCMs (hiCMs) at an efficiency of 40%-60%, approximately double that of previous protocols, within just 2 weeks. The resulting hiCMs display cardiomyocyte-like sarcomere structure, gene expression, and calcium oscillation. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2019).

Early transcriptional changes in cardiac mitochondria during chronic doxorubicin exposure and mitigation by dexrazoxane in mice.

Identification of early biomarkers of cardiotoxicity could help initiate means to ameliorate the cardiotoxic actions of clinically useful drugs such as doxorubicin (DOX). Since DOX has been shown to target mitochondria, transcriptional levels of mitochondria-related genes were evaluated to identify early candidate biomarkers in hearts of male B6C3F1 mice given a weekly intravenous dose of 3mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, 12, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice was pretreated (intraperitoneally) with the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg) 30 min before each weekly dose of DOX or SAL. At necropsy a week after the last dose, increased plasma concentrations of cardiac troponin T (cTnT) were detected at 18 and 24 mg/kg cumulative DOX doses, whereas myocardial alterations were observed only at the 24 mg/kg dose. Of 1019 genes interrogated, 185, 109, 140, 184, and 451 genes were differentially expressed at 6, 9, 12, 18, and 24 mg/kg cumulative DOX doses, respectively, compared to concurrent SAL-treated controls. Of these, expression of 61 genes associated with energy metabolism and apoptosis was significantly altered before and after occurrence of myocardial injury, suggesting these as early genomics markers of cardiotoxicity. Much of these DOX-induced transcriptional changes were attenuated by pretreatment of mice with DXZ. Also, DXZ treatment significantly reduced plasma cTnT concentration and completely ameliorated cardiac alterations induced by 24 mg/kg cumulative DOX. This information on early transcriptional changes during DOX treatment may be useful in designing cardioprotective strategies targeting mitochondria.

CELF4 Variant and Anthracycline-Related Cardiomyopathy: A Children's Oncology Group Genome-Wide Association Study.

PURPOSE: Interindividual variability in the dose-dependent association between anthracyclines and cardiomyopathy suggests that genetic susceptibility could play a role. The current study uses an agnostic approach to identify genetic variants that could modify cardiomyopathy risk. METHODS: A genome-wide association study was conducted in childhood cancer survivors with and without cardiomyopathy (cases and controls, respectively). Single-nucleotide polymorphisms (SNPs) that surpassed a prespecified threshold for statistical significance were independently replicated. The possible mechanistic significance of validated SNP(s) was sought by using healthy heart samples. RESULTS: No SNP was marginally associated with cardiomyopathy. However, SNP rs1786814 on the CELF4 gene passed the significance cutoff for gene-environment interaction (Pge = 1.14 × 10(-5)). Multivariable analyses adjusted for age at cancer diagnosis, sex, anthracycline dose, and chest radiation revealed that, among patients with the A allele, cardiomyopathy was infrequent and not dose related. However, among those exposed to greater than 300 mg/m(2) of anthracyclines, the rs1786814 GG genotype conferred a 10.2-fold (95% CI, 3.8- to 27.3-fold; P < .001) increased risk of cardiomyopathy compared with those who had GA/AA genotypes and anthracycline exposure of 300 mg/m(2) or less. This gene-environment interaction was successfully replicated in an independent set of anthracycline-related cardiomyopathy. CUG-BP and ETR-3-like factor proteins control developmentally regulated splicing of TNNT2, the gene that encodes for cardiac troponin T (cTnT), a biomarker of myocardial injury. Coexistence of more than one cTnT variant results in a temporally split myofilament response to calcium, which causes decreased contractility. Analysis of TNNT2 splicing variants in healthy human hearts suggested an association between the rs1786814 GG genotype and coexistence of more than one TNNT2 splicing variant (90.5% GG v 41.7% GA/AA; P = .005). CONCLUSION: We report a modifying effect of a polymorphism of CELF4 (rs1786814) on the dose-dependent association between anthracyclines and cardiomyopathy, which possibly occurs through a pathway that involves the expression of abnormally spliced TNNT2 variants.

TNNT1, TNNT2, and TNNT3: Isoform genes, regulation, and structure-function relationships.

Troponin T (TnT) is a central player in the calcium regulation of actin thin filament function and is essential for the contraction of striated muscles. Three homologous genes have evolved in vertebrates to encode three muscle type-specific TnT isoforms: TNNT1 for slow skeletal muscle TnT, TNNT2 for cardiac muscle TnT, and TNNT3 for fast skeletal muscle TnT. Alternative splicing and posttranslational modifications confer additional structural and functional variations of TnT during development and muscle adaptation to various physiological and pathological conditions. This review focuses on the TnT isoform genes and their molecular evolution, alternative splicing, developmental regulation, structure-function relationships of TnT proteins, posttranslational modifications, and myopathic mutations and abnormal splicing. The goal is to provide a concise summary of the current knowledge and some perspectives for future research and translational applications.

Circulating Histone Concentrations Differentially Affect the Predominance of Left or Right Ventricular Dysfunction in Critical Illness.

OBJECTIVES: Cardiac complications are common in critical illness and associated with grave consequences. In this setting, elevated circulating histone levels have been linked to cardiac injury and dysfunction in experimental models and patients with sepsis. The mechanisms underlying histone-induced cardiotoxicity and the functional consequences on left ventricle and right ventricle remain unclear. This study aims to examine dose-dependent effects of circulating histones on left ventricle and right ventricle function at clinically relevant concentrations. DESIGN: Prospective laboratory study with in vitro and in vivo investigations. SETTING: University research laboratory. SUBJECTS: Twelve-week old male C57BL/6N mice. INTERVENTIONS: Cultured cardiomyocytes were incubated with clinically relevant histone concentrations, and a histone infusion mouse model was also used with hemodynamic changes characterized by echocardiography and left ventricle/right ventricle catheter-derived variables. Circulating histones and cardiac troponin levels were obtained from serial blood samples. MEASUREMENTS AND MAIN RESULTS: IV histone infusion caused time-dependent cardiac troponin elevation to indicate cardiac injury. At moderate sublethal histone doses (30 mg/kg), left ventricular contractile dysfunction was the prominent abnormality with reduced ejection fraction and prolonged relaxation time. At high doses (≥ 60 mg/kg), pulmonary vascular obstruction induced right ventricular pressure increase and dilatation, but left ventricular end-diastolic volume improved because of reduced blood return from the lungs. Mechanistically, histones induced profound calcium influx and overload in cultured cardiomyocytes with dose-dependent detrimental effects on intracellular calcium transient amplitude, contractility, and rhythm, suggesting that histones directly affect cardiomyocyte function adversely. However, increasing histone-induced neutrophil congestion, neutrophil extracellular trap formation, and thrombosis in the pulmonary microvasculature culminated in right ventricular dysfunction. Antihistone antibody treatment abrogated histone cardiotoxicity. CONCLUSIONS: Circulating histones significantly compromise left ventricular and right ventricular function through different mechanisms that are dependent on histone concentrations. This provides a translational basis to explain and target the spectral manifestations of cardiac dysfunction in critical illness.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction.

High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.

Silencing of nodal modulator 1 inhibits the differentiation of P19 cells into cardiomyocytes.

Nodal modulator 1 (NOMO1), a highly conserved transmembrane protein, has been identified as a part of a protein complex that participates in the Nodal signaling pathway, a critical determinant of heart and visceral organ formation. We previously found that the NOMO1 gene was substantially downregulated in human ventricular septal defect (VSD) myocardium and, thus, may be an important molecular pathway in human heart development. In this study, we aimed to investigate the effects of NOMO1 gene silencing by RNA interference (RNAi) during early mouse cardiac differentiation using P19 cells as a model system. Our results revealed that the differentiated P19 cell population exhibited downregulated NOMO1 levels and expressed lower levels of Nodal signaling mediators, such as Nodal, Cripto and Smad2, than the negative control. Similarly, cardiomyocyte-specific sarcomeric markers, such as cardiac troponin T, as well as expression of cardiogenesis-related transcriptional factors, such as Nkx2.5, Gata4 and Tbx5 were found to be downregulated in P19 differentiated cardiomyocytes in NOMO1-silenced cells when compared to controls. In conclusion, our results indicate that NOMO1 gene knockdown inhibits the differentiation of P19 cells into cardiomyocytes, which highlights a potential role for NOMO1 in early cardiogenesis.

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.

NH2-terminal truncations of cardiac troponin I and cardiac troponin T produce distinct effects on contractility and calcium homeostasis in adult cardiomyocytes.

Cardiac troponin I (TnI) has an NH2-terminal extension that is an adult heart-specific regulatory structure. Restrictive proteolytic truncation of the NH2-terminal extension of cardiac TnI occurs in normal hearts and is upregulated in cardiac adaptation to hemodynamic stress or ß-adrenergic deficiency. NH2-terminal truncated cardiac TnI (cTnI-ND) alters the conformation of the core structure of cardiac TnI similarly to that produced by PKA phosphorylation of Ser(23/24) in the NH2-terminal extension. At organ level, cTnI-ND enhances ventricular diastolic function. The NH2-terminal region of cardiac troponin T (TnT) is another regulatory structure that can be selectively cleaved via restrictive proteolysis. Structural variations in the NH2-terminal region of TnT also alter the molecular conformation and function. Transgenic mouse hearts expressing NH2-terminal truncated cardiac TnT (cTnT-ND) showed slower contractile velocity to prolong ventricular rapid-ejection time, resulting in higher stroke volume. Our present study compared the effects of cTnI-ND and cTnT-ND in cardiomyocytes isolated from transgenic mice on cellular morphology, contractility, and calcium kinetics. Resting cTnI-ND, but not cTnT-ND, cardiomyocytes had shorter length than wild-type cells with no change in sarcomere length. cTnI-ND, but not cTnT-ND, cardiomyocytes produced higher contractile amplitude and faster shortening and relengthening velocities in the absence of external load than wild-type controls. Although the baseline and peak levels of cytosolic Ca(2+) were not changed, Ca(2+) resequestration was faster in both cTnI-ND and cTnT-ND cardiomyocytes than in wild-type control. The distinct effects of cTnI-ND and cTnT-ND demonstrate their roles in selectively modulating diastolic or systolic functions of the heart.

Serelaxin: a novel therapy for acute heart failure with a range of hemodynamic and non-hemodynamic actions.

Acute heart failure (AHF) is characterized by high morbidity and mortality and high costs. Although the treatment of AHF has not changed substantially in recent decades, it is becoming clear that treatment strategies for AHF need to address both the immediate hemodynamic abnormalities giving rise to congestion as well as prevent organ damage that can influence long-term prognosis. Serelaxin, the recombinant form of human relaxin-2, a naturally occurring peptide hormone, has been found to significantly improve symptoms and signs of AHF, prevent in-hospital worsening heart failure, as well as significantly improve 180-day cardiovascular and all-cause mortality after a 48-h infusion commenced within 16 h of presentation (RELAX-AHF study). Available data suggest that the clinical benefits may be attributable to a potential combination of multiple actions of serelaxin, including improving systemic, cardiac, and renal hemodynamics, and protecting cells and organs from damage via anti-inflammatory, anti-cell death, anti-fibrotic, anti-hypertrophic, and pro-angiogenic effects. This manuscript describes the short- and long-term effects of serelaxin in AHF patients, analyzing how these effects can be explained by taking into account the range of hemodynamic and non-hemodynamic actions of serelaxin. In addition, this paper also addresses several aspects related to the role of serelaxin in the therapy of AHF that remain to be clarified and warrant further investigation.

Human slow troponin T (TNNT1) pre-mRNA alternative splicing is an indicator of skeletal muscle response to resistance exercise in older adults.

Slow skeletal muscle troponin T (TNNT1) pre-messenger RNA alternative splicing (AS) provides transcript diversity and increases the variety of proteins the gene encodes. Here, we identified three major TNNT1 splicing patterns (AS1-3), quantified their expression in the vastus lateralis muscle of older adults, and demonstrated that resistance training modifies their relative abundance; specifically, upregulating AS1 and downregulating AS2 and AS3. In addition, abundance of TNNT1 AS2 correlated negatively with single muscle fiber-specific force after resistance training, while abundance of AS1 correlated negatively with V max. We propose that TNNT1 AS1, AS2 and the AS1/AS2 ratio are potential quantitative biomarkers of skeletal muscle adaptation to resistance training in older adults, and that their profile reflects enhanced single fiber muscle force in the absence of significant increases in fiber cross-sectional area.

Doxorubicin has in vivo toxicological effects on ex vivo cultured mesenchymal stem cells.

Doxorubicin (dox) is an effective chemotherapeutic agent that leads to cardiotoxicity. An alternative treatment for dox-cardiotoxicity is autologous mesenchymal stem cells (MSCs) transplantation. It remains unclear if dox has deleterious effects on MSCs from subjects under chemotherapy, therefore this study aimed to evaluate dox in vivo toxicological effects on ex vivo cultured MSCs, inferring whether autologous transplantation may be an alternative treatment in patients who are exposed to the drug. Wistar rats received either dox or saline. Following treatments, animals were sacrificed and bone marrow MSCs were isolated, characterized for cell surface markers and assessed according to their viability, alkaline phosphatase production, and proliferation kinetics. Moreover, MSCs were primed to cardiac differentiation and troponin T and connexin 43 expressions were evaluated. Compared to control, undifferentiated MSCs from dox group kept the pattern for surface marker and had similar viability results. In contrast, they showed lower alkaline phosphatase production, proliferation rate, and connexin 43 expression. Primed MSCs from dox group showed lower troponin T levels. It was demonstrated a toxic effect of dox in host MSCs. This result renders the possibility of autologous MSCs transplantation to treat dox-cardiotoxicity, which could be a non-suitable option for subjects receiving such antineoplastic agent.

Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles.

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and ß-Tm) and slow (γ-Tm and ß-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, ß and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Characteristics of mesenchymal stem cells isolated from bone marrow of giant panda.

In present study, we report on bone marrow (BM) mesenchymal stem cells (MSCs) that are isolated from giant pandas. Cells were collected from the BM of two stillborn giant pandas. The cells were cultured and expanded in 10% fetal bovine serum medium. Cell morphology was observed under an inverted microscopy, and the proliferation potential of the cells was evaluated by counting cell numbers for eight consecutive days. Differentiation potentials of the cells were determined by using a variety of differentiation protocols for osteocytes, adipocytes, neuron cells, and cardiomyocytes. Meanwhile, the specific gene expressions for MSCs or differentiated cells were analyzed by RT-PCR. The isolated cells exhibited a fibroblast-like morphology; expressed mesenchymal specific markers such as cluster of differentiation 73 (CD73), SRY (sex determining region Y)-box 2 (SOX-2), guanine nucleotide-binding protein-like 3 (GNL3), and stem cell factor receptor (SCFR); and could be differentiated into osteocytes and adipocytes that were characterized by Alizarin Red and Oil Red O staining. Under appropriate induction conditions, these cells were also able to differentiate into neuroglial-like or myocardial-like cells that expressed specific myocardial markers such as GATA transcription factors 4 (GATA-4), cardiac troponin T (cTnT), and myosin heavy chain 7B (MYH7B), or neural specific markers such as Nestin and glial fibrillary acidic protein (GFAP). This study demonstrated stem cells recovery and growth from giant pandas. The findings suggest that cells isolated from the BM of giant pandas have a high proliferative capacity and multiple differentiation potential in vitro which might aid conservation efforts.

Expression of cardiac proteins in neonatal cardiomyocytes on PGS/fibrinogen core/shell substrate for Cardiac tissue engineering.

BACKGROUND: Heart failure due to myocardial infarction remains the leading cause of death worldwide owing to the inability of myocardial tissue regeneration. The aim of this study is to develop a core/shell fibrous cardiac patch having desirable mechanical properties and biocompatibility to engineer the infarcted myocardium. METHOD: We fabricated poly(glycerol sebacate)/fibrinogen (PGS/fibrinogen) core/shell fibers with core as elastomeric PGS provides suitable mechanical properties comparable to that of native tissue and shell as fibrinogen to promote cell-biomaterial interactions. The PGS/fibrinogen core/shell fibers and fibrinogen nanofibers were characterized by SEM, contact angle and tensile testing to analyze the fiber morphology, wettability, and mechanical properties of the scaffold. The cell-scaffold interactions were analyzed using isolated neonatal cardiomyocytes for cell proliferation, confocal analysis for the expression of marker proteins α-actinin, Troponin-T, ß-myosin heavy chain and connexin 43 and SEM analysis for cell morphology. RESULTS: We observed PGS/fibrinogen core/shell fibers had a Young's modulus of about 3.28 ± 1.7 MPa, which was comparable to that of native myocardium. Neonatal cardiomyocytes cultured on these scaffolds showed normal expression of cardiac specific marker proteins α-actinin, Troponin, ß-myosin heavy chain and connexin 43 to prove PGS/fibrinogen core/shell fibers have potential for cardiac tissue engineering. CONCLUSION: Results indicated that neonatal cardiomyocytes formed predominant gap junctions and expressed cardiac specific marker proteins on PGS/fibrinogen core/shell fibers compared to fibrinogen nanofibers, indicating PGS/fibrinogen core/shell fibers may serve as a suitable cardiac patch for the regeneration of infarcted myocardium.

Minor myocardial damage is a prevalent condition in patients with acute heart failure syndromes and preserved systolic function with long-term prognostic implications: a report from the CIAST-HF (Collaborative Italo-Argentinean Study on cardiac Troponin T in Heart Failure) study.

BACKGROUND: Half of patients with acute heart failure syndromes (AHFS) have preserved left ventricular ejection fraction (PLVEF). In this setting, the role of minor myocardial damage (MMD), as identified by cardiac troponin T (cTnT), remains to be established. AIM: To evaluate the prevalence and long-term prognostic significance of cTnT elevations in patients with AHFS and PLVEF. PATIENTS AND METHODS: This retrospective, multicenter, collaborative study included 500 patients hospitalized for AHFS with PLVEF (ejection fraction ≥40%) between October 2000 and December 2006. Blood samples were collected within 12 hours after admission and were assayed for cTnT. MMD was defined as a cTnT value of ≥0.020 ng/mL. RESULTS: Mean age was 73 ± 12 years, 47% were female, 38% had an ischemic etiology, and New York Heart Association (NYHA) class was 2.2 ± 0.7. Mean cTnT value was 0.149 ± 0.484 ng/mL, and cTnT was directly correlated with serum creatinine (Spearman's Rho = 0.35, P < .001) and NYHA class (0.25, P < .001). MMD was diagnosed in 220 patients (44%). Patients with MMD showed lower left ventricular ejection fraction (P < .05), higher serum creatinine (P < .001), higher prevalence of ischemic etiology and diabetes mellitus, a worse NYHA class (P < .001), and higher natriuretic peptide levels (P < .001) as compared with patients without MMD. At 6-month follow-up, overall event-free survival was 55% and 75% in patients with and without MMD (P < .001), respectively. On multivariate Cox regression analysis, only NYHA class (HR = 1.50; P = .002) and MMD (HR = 1.81; P = .001) were identified as predictors of events. CONCLUSIONS: Increased cTnT levels were detected in approximately 50% of patients with AHFS with preserved systolic function, and were found to correlate with clinical measures of disease severity. The presence of MMD was associated with a worse long-term outcome, lending support to cTnT-based risk stratification in the setting of AHFS.

Frequency, determinants and outcome of elevated troponin in acute ischemic stroke patients.

BACKGROUND: Myocardial injury indicated by elevation of cardiac troponins (cTnT) can be observed in acute ischemic stroke patients. Frequency, determinants and prognostic value are still unsettled. METHODS: We performed a retrospective analysis including all consecutive ischemic stroke patients admitted to our stroke unit within 72 h after symptom onset in a one-year period. Multivariable logistic regression analyses were conducted to identify determinants of cTnT elevation and to detect factors independently associated with unfavorable short-term outcome (modified Rankin scale >2), major neurologic improvement (improvement of NIHSS> =8 or NIHSS 0-1) and in-hospital mortality. RESULTS: Admission cTnT levels were measured in 715 ischemic stroke patients. Frequency of cTnT elevation was 14% (n=103). Factors independently associated with increased cTnT were higher stroke severity (p=0.04), renal insufficiency (p<0.001), pre-existing coronary artery disease (p=0.03), hypercholesterolemia (p=0.02) and insular cortex involvement (p<0.001). After exclusion of patients with renal insufficiency and coronary artery disease frequency of cTnT elevation was 10% (n=44) and only insular cortex involvement remained significantly associated. Increased cTnT on admission was an independent predictor of unfavorable outcome (adjusted odds ratio 2.65 [95% confidence interval 1.29-5.46]) and in-hospital mortality (4.51 [1.93-10.57]). There was a trend towards a negative association of cTnT elevation with major neurologic improvement (0.54 [0.27-1.07]). CONCLUSIONS: Elevation of cTnT occurs in every seventh patient with acute ischemic stroke and is independently associated with poor short-term outcome and mortality. Patients with strokes affecting the insular cortex are particularly prone to myocardial injury justifying intensive cardiac monitoring.

Overexpression of Csx/Nkx2.5 and GATA-4 enhances the efficacy of mesenchymal stem cell transplantation after myocardial infarction.

BACKGROUND: The high death rate of the transplanted stem cells in the infarcted heart and low efficiency of differentiation toward cardiomyocytes show that mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) is not effective. Csx/Nkx2.5 and GATA-4 are considered to be key regulators of cardiogenesis. The aim of the present study was to investigate the effect of transplanting MSC overexpressing Csx/Nkx2.5 and GATA-4 (MSCs-CG) after MI. METHODS AND RESULTS: According to acridine orange/ethidium bromide staining, MSCs-CG were more resistant to anoxia as compared with MSCs in vitro. In a mouse MI model, ejection fraction and fractional shortening were higher in the MSC-CG group than in the MSC or phosphate-buffered saline group. Wall thickness of the infarct area was increased and collagen deposition was clearly reduced in the MSC-CG group as compared with the other groups. There were more surviving MSCs in the MSC-CG group than in the MSC group. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). Cx-43 was localized between Y chromosome-positive cells and recipient cardiomyocytes. Microvessel density in the peri-infarct regions and infarct regions increased significantly in the MSC-CG group. CONCLUSIONS: Transplantation of MSCs overexpressing Csx/Nkx2.5 and GATA-4 represents a new treatment strategy with the potential to improve cardiac function after MI.

Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test.

The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.