Cardiomyocyte Remodeling Research Articles

Stilbenoid pterostilbene complexed with cyclodextrin preserves left ventricular function after myocardial infarction in rats: possible involvement of thiol proteins and modulation of phosphorylated GSK-3β

Oxidative stress alters signalling pathways for survival and cell death favouring the adverse remodelling of postmyocardial remnant cardiomyocytes, promoting functional impairment. The administration of pterostilbene (PTS), a phytophenol with antioxidant potential, can promote cardioprotection and represents a therapeutic alternative in acute myocardial infarction (AMI). The present study aims to explore the effects of oral administration of PTS complexed with hydroxypropyl-β-cyclodextrin HPβCD (PTS:HPβCD complex) on the glutathione cycle, thiol protein activities and signalling pathways involving the protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) proteins in the left ventricle (LV) of infarcted rats. Animals were submitted to acute myocardial infarction through surgical ligation of the descending anterior branch of the left coronary artery and received over 8 days, by gavage, PTS:HPβCD complex at dose of 100 mg kg−1 day−1 (AMI + PTS group) or vehicle (aqueous solution with HPβCD) divided into Sham-operated (SHAM) and infarcted (AMI) groups. The results showed that the PBS: HPβCD complex decreased lipid peroxidation, prevented the decrease in thioredoxin reductase (TRxR) activity, and increased the activity of glutathione-S-transferase (GST) and glutaredoxin (GRx). Additionally, the expression of nuclear factor-erythroid two (Nrf2) and p-GSK-3β was increased, whereas the p-GSK-3β/GSK-3β ratio was reduced in the LV of the infarcted animals. Overall, the PTS:HPβCD complex modulates activity of thiol-dependent enzymes and induces to the expression of antioxidant proteins, improving systolic function and mitigating the adverse cardiac remodelling post infarction.

Phosphodiesterase-5 Is Elevated in Failing Single Ventricle Myocardium and Affects Cardiomyocyte Remodeling In Vitro.

Background Single ventricle (SV) congenital heart disease is fatal without intervention, and eventual heart failure is a major cause of morbidity and mortality. Although there are no proven medical therapies for the treatment or prevention of heart failure in the SV heart disease population, phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are increasingly used. Although the pulmonary vasculature is the primary target of PDE5i therapy in patients with SV heart disease, the effects of PDE5i on the SV heart disease myocardium remain largely unknown. We sought to determine PDE5 expression and activity in the single right ventricle of SV heart disease patients relative to nonfailing controls and to determine whether PDE5 impacts cardiomyocyte remodeling using a novel serum-based in vitro model. Methods and Results PDE5 expression (n=9 nonfailing; n=7 SV heart disease), activity (n=8 nonfailing; n=9 SV heart disease), and localization (n=3 SV heart disease) were determined in explanted human right ventricle myocardium. PDE5 is expressed in SV heart disease cardiomyocytes, and PDE5 protein expression and activity are increased in SV heart disease right ventricle compared with nonfailing right ventricle. Isolated neonatal rat ventricular myocytes were treated for 72 hours with nonfailing or SV heart disease patient serum±sildenafil. Reverse transcription quantitative polymerase chain reaction (n=5 nonfailing; n=12 SV heart disease) and RNA sequencing (n=3 nonfailing; n=3 SV heart disease) were performed on serum-treated neonatal rat ventricular myocytes and demonstrated that treatment with SV heart disease sera results in pathological gene expression changes that are attenuated with PDE5i. Conclusions PDE5 is increased in failing SV heart disease myocardium, and pathological gene expression changes in SV heart disease serum-treated neonatal rat ventricular myocytes are abrogated by PDE5i. These results suggest that PDE5 represents an intriguing myocardial therapeutic target in this population.

NLRP3 deficiency accelerates pressure overload-induced cardiac remodeling via increased TLR4 expression.

NLRP3, a member of the nucleotide-binding oligomerization domain (NOD)-like receptor family, is involved in cardiac inflammation. However, the functional role of NLRP3 in cardiac remodeling is not clear. To investigate the roles of NLRP3 in pressure overload-induced cardiac remodeling, NLRP3 knockout and wild-type mice were subjected to aortic banding to induce cardiac remodeling. The data showed that NLRP3 expression was downregulated in the remodeling process. NLRP3 deficiency accelerated cardiac hypertrophy, fibrosis, and inflammation responses with deteriorating cardiac dysfunction in the pressure overload-induced cardiac remodeling mouse model. Neonatal rat cardiomyocytes were isolated and stimulated with phenylephrine (PE). We identified NLRP3 as a negative regulator of cardiomyocyte remodeling in PE-stimulated cardiomyocyte remodeling using adenovirus-NLRP3 and NLRP3 siRNA. Mechanistically, we found that the expression of Toll-like receptor (TLR) 4 was upregulated in NLRP3-deficient mouse hearts and PE-stimulated cardiomyocytes. NLRP3 knockout mice subjected to a TLR4 inhibitor revealed a relieved cardiac remodeling response with improved cardiac dysfunction. Our data suggested that NLRP3 could be a therapeutic target for cardiac remodeling and heart failure. KEY MESSAGES: NLRP3 expression was downregulated in the remodeling process. NLRP3 deficiency accelerated pressure overload-induced cardiac remodeling. NLRP3 acted as a negative regulator of cardiomyocyte remodeling via downregulating TLR4.

Abstract 260: Adverse Transverse-Tubule Remodeling in a Rat Model of Heart Failure is Attenuated by Triiodothyronine (T3) Treatment

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-MI cardiomyocyte remodeling by maintaining T-tubule (TT) organization, thus improving contractility. Methods: Coronary artery ligation (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5ug/kg/d) or vehicle in drinking water for 16 wks (10/gp). After echocardiographic/hemodynamic analyses, ventricular myocytes were isolated by collagenase digestion and plated on laminin-coated glass coverslips for TT analysis or analyzed for contractility using IonOptix’s edge-detection method. Live cell staining with wheat germ agglutinin (WGA)-488 or di-8-ANNEPS labeled sarcolemma and TT, and multiple z-stack images were captured by scanning confocal microscopy and TT organization measured using TTorg program. Ventricular RNA was analyzed by qPCR. Results: Echocardiography and in vivo hemodynamic measurements showed significantly improved systolic and diastolic function in T3- vs vehicle-treated MI rats. IonOpix analysis of isolated ventricular myocytes showed significant dysfunction in measures of relaxation in MI myocytes compared to sham, and improvements with T3: max relaxation velocity (Vmax), 2.98±1.41 vs 1.59±0.33, p<0.05; time to Vmax (sec), 0.23±0.04 vs 0.31±0.02, p<0.001, MI+T3 vs MI). Similarly, time to peak contraction was shortened by T3 treatment (0.16±0.02 vs 0.20±0.01 sec., MI+T3 vs MI; p<0.01. TT power measured by TTorg analysis of WGA-488 or di-8-ANNEPS labeled myocytes showed significant disorganization of the TT system in MI myocytes that was normalized by T3-treatment (TTpower = 82±7 sham, 74±8 MI and 79±6 MI+T3; sham vs MI p<0.0001; MI vs MI+T3, p<0.0001). qPCR results indicated that reduced expression of Bin1, Jph2, RyR2 and LTCC in the MI myocardium was normalized by T3. In conclusion, T3 treatment prevented TT disorganization in the post-MI cardiomyocyte, normalized contractile and relaxation parameters and improved cardiac output, thus supporting the therapeutic potential of T3 in heart failure.

Heat shock protein inducer reverses the contractile and structural remodeling of HL-1 cardiomyocytes in experimental model of Atrial Fibrillation

Background: Atrial Fibrillation (AF) is the most common tachyarrhythmia associated with electrical, structural remodeling and contractile dysfunction, which is rooted in an impaired protein quality control (PQC) system. PQC regulates the correct folding, trafficking and degradation of proteins in cells. Heat shock proteins (HSPs) play a critical role in facilitating the PQC. Our previous studies showed that the HSP inducers geranylgeranylaceton (GGA) and its derivatives prevent contractile dysfunction in tachypaced atrial cardiomyocyte and Drosophila model for AF. Here we tested whether the potent HSP inducer GGA59 also restores contractile dysfunction in tachypaced cardiomyocytes and explored the underlying mechanisms.

At the heart of inter- and intracellular signaling: the intercalated disc

Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.

Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction Is Distinctive-A New Preclinical Model.

BackgroundAmong the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need—and the lack of HFpEF‐specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology.Methods and ResultsThe selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%–90% control) with marked diastolic dysfunction (increased E/E′). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L‐type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed.ConclusionsThus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF—and suggest a basis for new therapeutic strategies.

The Transcription Factor ATF4 Promotes Expression of Cell Stress Genes and Cardiomyocyte Death in a Cellular Model of Atrial Fibrillation.

Introduction Cardiomyocyte remodelling in atrial fibrillation (AF) has been associated with both oxidative stress and endoplasmic reticulum (ER) stress and is accompanied by a complex transcriptional regulation. Here, we investigated the role the oxidative stress and ER stress responsive bZIP transcription factor ATF4 plays in atrial cardiomyocyte viability and AF induced gene expression. Methods HL-1 cardiomyocytes were subjected to rapid field stimulation. Forced expression of ATF4 was achieved by adenoviral gene transfer. Using global gene expression analysis and chromatin immunoprecipitation, ATF4 dependent transcriptional regulation was studied, and tissue specimen of AF patients was analysed by immunohistochemistry. Results Oxidative stress and ER stress caused a significant reduction in cardiomyocyte viability and were associated with an induction of ATF4. Accordingly, ATF4 was also induced by rapid field stimulation mimicking AF. Forced expression of wild type ATF4 promoted cardiomyocyte death. ATF4 was demonstrated to bind to the promoters of several cell stress genes and to induce the expression of a number of ATF4 dependent stress responsive genes. Moreover, immunohistochemical analyses showed that ATF4 is expressed in the nuclei of cardiomyocytes of tissue specimen obtained from AF patients. Conclusion ATF4 is expressed in human atrial cardiomyocytes and is induced in response to different types of cell stress. High rate electrical field stimulation seems to result in ATF4 induction, and forced expression of ATF4 reduces cardiomyocyte viability.

The MEF2 transcriptional target DMPK induces loss of sarcomere structure and cardiomyopathy

The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure.

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy

BackgroundRight ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca2+ remodeling. Methods and resultsAfter PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca2+]i transients and increased sarcoplasmic reticulum (SR) Ca2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser16-phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca2+-ATPase) pump abundance. Moreover, after PH induction, Ca2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca2+-release-activated Ca2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca2+]i transients amplitude, the SR Ca2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. ConclusionsThese new findings demonstrate RV-specific cellular Ca2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca2+ current participates in this Ca2+ remodeling in RVH secondary to PH.

TNF‐α Mediated β1 Integrin Inactivation is a Novel Mechanism Mediating Left Ventricular Dilatation

OBJECTIVEWe previously reported that β1 integrin‐mediated adhesion of cardiac myocytes to components of the extracellular matrix (ECM) is significantly reduced in the aortocaval fistula model of chronic volume overload. Other studies have shown that myocardial TNF‐α is significantly elevated in this model. Therefore, this study sought to test the hypothesis that pathophysiologic levels of TNF‐α produce ventricular dilatation by significantly reducing integrin‐mediated adhesion of cardiomyocytes to the ECM.METHODSTo this end, adult male Sprague‐Dawley rats (n=10) were continuously infused with TNF‐α (2.5 μg/kg/hr) via subcutaneously implanted osmotic minipumps (Alzet model 2002, Alza Corp, Mountain View, CA) for 3 days. This rate of infusion resulted in systemic levels of biologically active TNF‐α of 17 ± 6.3 pg/ml of serum, which is comparable to that reported in patients with heart failure. Calcium tolerant cardiomyocytes were isolated from the TNF‐α infused rat hearts by perfusion with Joklik's MEM solution containing Type II collagenase (Worthington Biochemical Corp., Lakewood, NJ). The adhesion of these cardiomyocytes to laminin were compared to cardiomyocytes from a separate, age‐matched control group (n=7). The adhesion assay was carried out in 24‐well culture plates coated with laminin (Becton‐Dickinson; Bedford, MA) incubated at room temperature for 1 hr. In a subsequent study the effect of a continuous TNF‐α infusion on the left ventricular pressure‐volume relationships in normal hearts (n=3) was determined using a blood perfused Langendorff isolated heart preparation prior to (baseline) and 30 minutes after initiating the TNF‐α infusion. Western blots were performed with primary antibodies directed against phosphospecific Src kinase tyrosine residues (pY418) and (pY529).RESULTSCardiomyocyte adhesion to laminin in the TNF‐α infused rats was significantly decreased relative to the untreated controls (27.1 ± 3.2% vs 40.3 ± 3.0%, respectively; p<0.05). As can be seen in Figure 1, perfusion with TNF‐α produced rapid left ventricular dilatation comparable to that previously reported after β1 integrin inactivation. The significant right shift in the pressure‐volume relationship induced by TNF‐α reflects the rapid development of ventricular dilatation. Figure 2 demonstrates TNF‐α infusion produces contrasting effects of significant tyrosine Y418 phosphorylation (Panel A) and dephosphorylation of tyrosine Y529.CONCLUSIONTNF‐α produces a marked decrease in adult cardiomyocyte adhesion to the ECM; and TNF‐α mediates acute alterations in integrin‐mediated cardiomyocyte adhesion which involves Src kinase‐mediated integrin inactivation. These finding implicate an acute TNF‐α induced reduction in cardiomyocyte integrin function as a novel mechanism contributing to ventricular dilatation. TNF‐α mediated changes in mechanotransduction also represent a likely mechanism responsible for gender differences in cardiomyocyte remodeling previously reported in the aortocaval fistula model.Support or Funding InformationAmerican Heart Association Southern Research Consortium Grant‐In‐Aid 0051505BThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exosomes from Suxiao Jiuxin pill-treated cardiac mesenchymal stem cells decrease H3K27 demethylase UTX expression in mouse cardiomyocytes in vitro.

Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine for the treatment of acute coronary syndrome in China, which contains two principal components, tetramethylpyrazine (TMP) and borneol (BOR). Thus far, however, the molecular mechanisms underlying the beneficial effects of SJP on the cardiac microenvironment are unknown. Cardiac mesenchymal stem cells (C-MSCs) communicate with cardiomyocytes (CMs) through the release of microvesicles (exosomes) to restore cardiac homeostasis and elicit repair, in part through epigenetic regulatory mechanisms. In this study, we examined whether SJP treatment altered C-MSC-derived exosomes (SJP-Exos) to cause epigenetic chromatic remodeling in recipient CMs. C-MSC isolated from mouse hearts were pretreated with SJP (SJP-Exos), TMP (TMP-Exos) or BOR (BOR-Exos). Then, HL-1 cells, a mouse cardiomyocyte line, were treated with exosomes from control C-MSCs (Ctrl-Exos), SJP-Exos, TMP-Exos or BOR-Exos. Treatment with SJP-Exos significantly increased the protein levels of histone 3 lysine 27 trimethylation (H3K27me3), a key epigenetic chromatin marker for cardiac transcriptional suppression, in the HL-1 cells. To further explore the mechanisms of SJP-Exo-mediated H3K27me3 upregulation, we assessed the mRNA expression levels of key histone methylases (EZH1, EZH2 and EED) and demethylases (JMJD3 and UTX) in the exosome-treated HL-1 cells. Treatment with SJP-Exo selectively suppressed UTX expression in the recipient HL-1 cells. Furthermore, PCNA, an endogenous marker of cell replication, was significantly higher in SJP-Exo-treated HL-1 cells than in Ctrl-Exo-treated HL-1 cells. These results show that SJP-Exos increase cardiomyocyte proliferation and demonstrate that SJP can modulate C-MSC-derived exosomes to cause epigenetic chromatin remodeling in recipient cardiomyocytes; consequently, SJP-Exos might be used to promote cardiomyocyte proliferation.

The Unfolded Protein Response Contributes to Electrical Remodeling in Human Cardiomyocytes

The Unfolded Protein Response Contributes to Electrical Remodeling in Human Cardiomyocytes

Inhibition of Histone Deacetylases Induces K+ Channel Remodeling and Action Potential Prolongation in HL-1 Atrial Cardiomyocytes

Background/Aims: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. Methods: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K⁺ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. Results: Global HDAC inhibition increased histone acetylation and prolonged APD₉₀ in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K⁺ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K⁺ channel remodeling. Conclusion: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation.

The role of Midkine in Children with Dilated Cardiomyopathy

Introduction: While current medical therapies benefit adult heart failure (HF) patients, these therapies do not confer the same improvement in survival in children with HF. This difference suggests that the underlying cellular mechanisms in failing myocytes are uniquely regulated. We have found that serum circulating factors are important in cardiomyocyte remodelling in pediatric patients with Dilated Cardiomyopathy (DCM). In our preliminary results, we identified a specific heparin-binding growth factor, midkine (MDK), which is highly upregulated in the serum of pediatric DCM patients. The objective of this study is to investigate the relation between serum MDK levels and clinical outcome, and to define the effect of MDK in modulating pathologic responses. Methods and results: Our current serum bank contains 5 non-failing (NF) and 44 DCM samples from individual patients. The level of serum (circulating) MDK was determined in the banked serum from pediatric DCM patients by ELISA, and correlated with the patient’s clinical outcome. Our results show that MDK levels in serum was elevated in pediatric DCM patients who died or were transplanted in comparison with pediatric DCM patients who are clinically stable. The contribution of serum MDK to primary cardiomyocytes was investigated by treating neonatal rat ventricular myocytes (NRVMs) with human recombinant MDK for 72 hours. Our results show an increase in the expression of genes associated with pathologic remodelling in MDK-treated NRVMs. In addition, there is an increase in cell area and ANF expression in MDK-treated NRVMs compared to NF serum-treated cells. In our unique mouse model of pediatric cardiac pathologic growth (isoproterenol infusion –30mg/kg/day), MDK treatment (3mg/kg/week) resulted in increased heart/body weight. Conclusions: Our results suggest that MDK is detrimental to cardiac function. This finding combined with increased circulating MDK in end-stage pediatric DCM patients, suggests an important regulatory role for MDK in pediatric HF.

Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation.

BackgroundDerailment of proteostasis, the homeostasis of production, function, and breakdown of proteins, contributes importantly to the self‐perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in humans. Autophagy plays an important role in proteostasis by degrading aberrant proteins and organelles. Herein, we investigated the role of autophagy and its activation pathway in experimental and clinical AF.Methods and ResultsTachypacing of HL‐1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B‐II expression, autophagic flux and autophagosome formation, and degradation of p62, resulting in reduction of Ca2+ amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4‐phenyl butyrate, overexpression of the ER chaperone‐protein heat shock protein A5, or overexpression of a phosphorylation‐blocked mutant of eukaryotic initiation factor 2α (eIF2α) prevents autophagy activation and Ca2+‐transient loss in tachypaced HL‐1 cardiomyocytes. Moreover, pharmacological inhibition of ER stress in tachypaced Drosophila confirms its role in derailing cardiomyocyte function. In vivo treatment with sodium salt of phenyl butyrate protected atrial‐tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L‐type Ca2+‐current reduction), cellular Ca2+‐handling/contractile dysfunction, and ER stress and autophagy; it also attenuated AF progression. Finally, atrial tissue from patients with persistent AF reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage.ConclusionsThese results identify ER stress–associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER‐stress inhibitor 4‐phenyl butyrate.

Experimentally induced intrauterine growth restriction in rabbits leads to differential remodelling of left versus right ventricular myocardial microstructure.

Intrauterine growth restriction (IUGR) is associated with foetal cardiac remodelling and dysfunction together with increased risk of cardiovascular disease in adulthood. Experimental data concerning effects of IUGR on cardiomyocyte and microvascularization anatomy are inconsistent and it is unknown whether both ventricles are similarly susceptible to in utero undersupply. Foetal IUGR was induced in pregnant rabbits at 25days of gestation by selective ligation of uteroplacental vessels. Foetal echocardiography showed systolic and diastolic dysfunction of both ventricles and body and heart weight were significantly reduced in response to IUGR. Design-based stereology revealed a decrease in cardiomyocyte number in both ventricles which was only in the left ventricle accompanied by a significantly higher cardiomyocyte mean volume. The proportion of mono- and bi-nucleated cardiomyocytes was unaltered between the groups indicating a similar maturation status. The number and length of cardiac capillaries in IUGR offspring was diminished in left but not in right ventricles. Foetal left and right ventricles are differently affected by placental insufficiency. While cardiomyocyte numbers are diminished in both ventricles, hypertrophic remodelling of cardiomyocytes and alterations in microvascularization is rather a left ventricular adaptation to IUGR. These unequal structural changes may be related to loading and developmental differences of the left and right ventricles.

Electro-mechanical conditioning of human iPSC-derived cardiomyocytes for translational research

RationaleImpaired maturation of human iPSC-derived cardiomyocytes (hiPSC-CMs) currently limits their use in experimental research and further optimization is required to unlock their full potential. ObjectiveTo push hiPSC-CMs towards maturation, we recapitulated the intrinsic cardiac properties by electro-mechanical stimulation and explored how these mimetic biophysical cues interplay and influence the cell behaviour. Methods and resultsWe introduced a novel device capable of applying synchronized electrical and mechanical stimuli to hiPSC-CM monolayers cultured on a PDMS membrane and evaluated effects of conditioning on cardiomyocyte structure and function. Human iPSC-CMs retained their cardiac phenotype and displayed adaptive structural responses to electrical (E), mechanical (M) and combined electro-mechanical (EM) stimuli, including enhanced membrane N-cadherin signal, stress-fiber formation and sarcomeric length shortening, most prominent under the EM stimulation. On the functional level, EM conditioning significantly reduced the transmembrane calcium current, resulting in a shift towards triangulation of intracellular calcium transients. In contrast, E and M stimulation applied independently increased the proportion of cells with L-Type calcium currents. In addition, calcium transients measured in the M-conditioned samples advanced to a more rectangular shape. ConclusionThe new methodology is a simple and elegant technique to systematically investigate and manipulate cardiomyocyte remodelling for translational applications. In the present study, we adjusted critical parameters to optimize a regimen for hiPSC-CM transformation. In the future, this technology will open up new avenues for electro-mechanical stimulation by allowing temporal and spatial control of stimuli which can be easily scaled up in complexity for cardiac development and disease modelling.

The protective role of small heat shock proteins in cardiac diseases: key role in atrial fibrillation

Atrial fibrillation (AF) is the most common tachyarrhythmia which is associated with increased morbidity and mortality. AF usually progresses from a self-terminating paroxysmal to persistent disease. It has been recognized that AF progression is driven by structural remodeling of cardiomyocytes, which results in electrical and contractile dysfunction of the atria. We recently showed that structural remodeling is rooted in derailment of proteostasis, i.e., homeostasis of protein production, function, and degradation. Since heat shock proteins (HSPs) play an important role in maintaining a healthy proteostasis, the role of HSPs was investigated in AF. It was found that especially small heat shock protein (HSPB) levels get exhausted in atrial tissue of patients with persistent AF and that genetic or pharmacological induction of HSPB protects against cardiomyocyte remodeling in experimental models for AF. In this review, we provide an overview of HSPBs as a potential therapeutic target for normalizing proteostasis and suppressing the substrates for AF progression in experimental and clinical AF and discuss HSP activators as a promising therapy to prevent AF onset and progression.

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase-induced cardiomyopathy: involvement of mitofusin-2.

Background and PurposeThe anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.Experimental ApproachNox2−/− and wild‐type (WT) littermate mice were administered DOX (12 mg·kg−1 over 3 weeks) prior to study at 4 weeks. Detailed mechanisms were investigated in murine HL‐1 cardiomyocytes, employing a robust model of oxidative stress, gene silencing and pharmacological tools.Key ResultsDOX‐induced cardiac dysfunction, cardiomyocyte remodelling, superoxide production and apoptosis in WT mice were attenuated in Nox2−/− mice. Transcriptional analysis of left ventricular tissue identified 152 differentially regulated genes (using adjusted P < 0.1) in DOX‐treated Nox2−/− versus WT mice, and network analysis highlighted ‘Cell death and survival’ as the biological function most significant to the dataset. The mitochondrial membrane protein, mitofusin‐2 (Mfn2), appeared as a strong candidate, with increased expression (1.5‐fold), confirmed by qPCR (1.3‐fold), matching clear published evidence of promotion of cardiomyocyte cell death. In HL‐1 cardiomyocytes, targeted siRNA knockdown of Nox2 decreased Mfn2 protein expression, but not vice versa. While inhibition of Nox2 activity along with DOX treatment attenuated its apoptotic and cytotoxic effects, reduced apoptosis after Mfn2 silencing reflected a sustained cytotoxic response and reduced cell viability.Conclusions and ImplicationsDOX‐induced and Nox2‐mediated up‐regulation of Mfn2, rather than contributing to cardiomyocyte dysfunction through apoptotic pathways, appears to promote a protective mechanism.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc