Adult Mouse Cardiac Myocytes Research Articles

Mutant SRF and YAP synthetic modified mRNAs drive cardiomyocyte nuclear replication

Introduction: Aging is associated with sarcopenia, myocyte loss, and dysfunction. The problem is compounded as the adult heart lacks the regenerative capacity to self-repair. Serum response factor’s (SRF’s) dual activity is essential for cell replication and heart cell differentiation. SRF interacts with cofactors, such as NKX2-5 and GATA4, which give cardiac-specific gene activity, and ETS factors such as ELK1 drive cell replication. Recently, the mutant YAP-5SA of the Hippo pathway was implicated in cardiomyocyte proliferation and growth. Aim: We hypothesized that disruption of interactions of SRF with NKX2-5 and GATA4 would lead to dedifferentiation of cardiomyocytes to a proliferative stem cell state and complement YAP-5SA to generate undifferentiated cardiomyocytes in a more primitive replicative state. Methods and results: To weaken SRF interactions with NKX2-5 and GATA4, alanine scanning mutations were generated across the SRF N-terminus of the MADS-box. One SRF mutant, SRF153(A3), was tested along with the YAP-5SA mutant, as degradable synthetic modified mRNAs (mmRNAs), in rat primary cardiomyocytes. To measure cell replication, adult cardiomyocytes were pulsed with alpha-EdU and then DAPI stained, while gene activity was assayed by RNA sequencing. To measure chromatin remodeling, Transposon 5 was used in ATAC sequencing. We observed that single and triple alanine substitutions of mutants centering over SRF-Lys154 essentially blocked myocyte differentiation, and NKX2-5 and GATA4 failed to stabilize mutated SRF DNA binding. Instead, many stem cell factors including NANOG and OCT4 were induced. SRF153(A3) does not recognize SRF response elements per ATAC sequencing and consequently induces stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. SRF153(A3) and YAP5SA mmRNA led to alpha-EDU incorporation in ~35% of the cardiomyocytes. DIAPH 3, a marker of the contractile ring during anaphase, appeared between and around replicated nuclei in three-month-old adult mouse cardiac myocytes. The combination of these synthetic mRNA increased nuclei replication with the expression of origin of replication genes, while genes associated with cardiomyocyte differentiation were down-regulated. ATAC sequencing revealed SRF153(A3) and YAP5SA mmRNA-induced chromatin remodeling of cell cycle, spindle, and growth factor genes by additive and synergistic activities. Conclusion: SRF153(A3) synthetic mmRNA and the mutant YAP-5SA mmRNA induced cardiomyocyte dedifferentiation, to nuclear replication in adult cardiac myocytes. The combinatorial use of mmRNA encoding SRF153(A3) and YAP-5SA has the potential to become a powerful clinical strategy for treating human heart disease.

Trans-cinnamaldehyde suppresses microtubule detyrosination and alleviates cardiac hypertrophy

BackgroundTrans-cinnamaldehyde (TCA) is a main compound of Cinnamomum cassia, used in traditional Chinese medicine to treat many ailments. Increasing evidence has demonstrated the therapeutic effects of TCA in cardiovascular diseases. PurposeThe present study aimed to determine whether TCA exerts antihypertrophic effects in vitro and in vivo and to elucidate the underlying mechanisms of these effects. MethodsNeonatal rat cardiac myocytes (NRCMs) and adult mouse cardiac myocytes (AMCMs) were treated with 50 μΜ phenylephrine (PE) for 48 h. Tubulin detyrosination, store-operated Ca2+ entry (SOCE), stromal interaction molecule-1 (STIM1)/Orai1 translocation, and calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathways were analyzed in NRCMs. Meanwhile, tubulin detyrosination, junctophilin-2, T-tubule distribution pattern, Ca2+ handling, and sarcomere shortening were observed in AMCMs. Male C57BL/6 mice were stimulated with PE (70 mg/kg per day) with or without TCA treatment for 2 weeks. Cardiac hypertrophy and tubulin detyrosination were also assessed. ResultsTCA was confirmed to alleviate cardiac hypertrophy induced by PE stimulation in vitro and in vivo. PE-induced cardiac hypertrophy was associated with excessive tubulin detyrosination and overexpression of vasohibin 1 (VASH1) and small vasohibin binding protein (SVBP), two key proteins responsible for tubulin detyrosination. These effects were largely blocked by TCA administration. PE treatment also enhanced SOCE with massive translocation of STIM1 and Orai1, Ca2+ mishandling, reduced sarcomere shortening, junctophilin-2, and T-tubule redistribution, all of which were significantly ameliorated by TCA administration. ConclusionOur study indicated that the therapeutic effects of TCA against cardiac hypertrophy may be associated with its ability to reduce tubulin detyrosination.

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

Branched chain amino acids exacerbate myocardial ischemia/reperfusion vulnerability via enhancing GCN2/ATF6/PPAR-α pathway-dependent fatty acid oxidation.

Rationale: Myocardial vulnerability to ischemia/reperfusion (I/R) injury is strictly regulated by energy substrate metabolism. Branched chain amino acids (BCAA), consisting of valine, leucine and isoleucine, are a group of essential amino acids that are highly oxidized in the heart. Elevated levels of BCAA have been implicated in the development of cardiovascular diseases; however, the role of BCAA in I/R process is not fully understood. The present study aims to determine how BCAA influence myocardial energy substrate metabolism and to further clarify the pathophysiological significance during cardiac I/R injury.Methods: Parameters of glucose and fatty acid metabolism were measured by seahorse metabolic flux analyzer in adult mouse cardiac myocytes with or without BCAA incubation. Chronic accumulation of BCAA was induced in mice receiving oral BCAA administration. A genetic mouse model with defective BCAA catabolism was also utilized. Mice were subjected to MI/R and the injury was assessed extensively at the whole-heart, cardiomyocyte, and molecular levels.Results: We confirmed that chronic accumulation of BCAA enhanced glycolysis and fatty acid oxidation (FAO) but suppressed glucose oxidation in adult mouse ventricular cardiomyocytes. Oral gavage of BCAA enhanced FAO in cardiac tissues, exacerbated lipid peroxidation toxicity and worsened myocardial vulnerability to I/R injury. Etomoxir, a specific inhibitor of FAO, rescued the deleterious effects of BCAA on I/R injury. Mechanistically, valine, leucine and their corresponding branched chain α-keto acid (BCKA) derivatives, but not isoleucine and its BCKA derivative, transcriptionally upregulated peroxisome proliferation-activated receptor alpha (PPAR-α). BCAA/BCKA induced PPAR-α upregulation through the general control nonderepresible-2 (GCN2)/ activating transcription factor-6 (ATF6) pathway. Finally, in a genetic mouse model with BCAA catabolic defects, chronic accumulation of BCAA increased FAO in myocardial tissues and sensitized the heart to I/R injury, which could be reversed by adenovirus-mediated PPAR-α silencing.Conclusions: We identify BCAA as an important nutrition regulator of myocardial fatty acid metabolism through transcriptional upregulation of PPAR-α. Chronic accumulation of BCAA, caused by either dietary or genetic factors, renders the heart vulnerable to I/R injury via exacerbating lipid peroxidation toxicity. These data support the notion that BCAA lowering methods might be potentially effective cardioprotective strategies, especially among patients with diseases characterized by elevated levels of BCAA, such as obesity and diabetes.

A Novel Role of Cyclic Nucleotide Phosphodiesterase 10A in Pathological Cardiac Remodeling and Dysfunction.

Heart failure is a leading cause of death worldwide. Cyclic nucleotide phosphodiesterases (PDEs), through degradation of cyclic nucleotides, play critical roles in cardiovascular biology and disease. Our preliminary screening studies have revealed PDE10A upregulation in the diseased heart. However, the roles of PDE10A in cardiovascular biology and disease are largely uncharacterized. The current study is aimed to investigate the regulation and function of PDE10A in cardiac cells and in the progression of cardiac remodeling and dysfunction. We used isolated adult mouse cardiac myocytes and fibroblasts, as well as preclinical mouse models of hypertrophy and heart failure. The PDE10A selective inhibitor TP-10, and global PDE10A knock out mice were used. We found that PDE10A expression remains relatively low in normal and exercised heart tissues. However, PDE10A is significantly upregulated in mouse and human failing hearts. In vitro, PDE10A deficiency or inhibiting PDE10A with selective inhibitor TP-10, attenuated cardiac myocyte pathological hypertrophy induced by Angiotensin II, phenylephrine, and isoproterenol, but did not affect cardiac myocyte physiological hypertrophy induced by IGF-1 (insulin-like growth factor 1). TP-10 also reduced TGF-β (transforming growth factor-β)-stimulated cardiac fibroblast activation, proliferation, migration and extracellular matrix synthesis. TP-10 treatment elevated both cAMP and cGMP levels in cardiac myocytes and cardiac fibroblasts, consistent with PDE10A as a cAMP/cGMP dual-specific PDE. In vivo, global PDE10A deficiency significantly attenuated myocardial hypertrophy, cardiac fibrosis, and dysfunction induced by chronic pressure overload via transverse aorta constriction or chronic neurohormonal stimulation via Angiotensin II infusion. Importantly, we demonstrated that the pharmacological effect of TP-10 is specifically through PDE10A inhibition. In addition, TP-10 is able to reverse pre-established cardiac hypertrophy and dysfunction. RNA-Sequencing and bioinformatics analysis further identified a PDE10A-regualted transcriptome involved in cardiac hypertrophy, fibrosis, and cardiomyopathy. Taken together, our study elucidates a novel role for PDE10A in the regulation of pathological cardiac remodeling and development of heart failure. Given that PDE10A has been proven to be a safe drug target, PDE10A inhibition may represent a novel therapeutic strategy for preventing and treating cardiac diseases associated with cardiac remodeling.

Potential Arrhythmogenic Role of TRPC Channels and Store-Operated Calcium Entry Mechanism in Mouse Ventricular Myocytes.

Background and Purpose: Store-operated calcium entry (SOCE) is an important physiological phenomenon that extensively mediates intracellular calcium ion (Ca2+) load. It has been previously found in myocytes isolated from neonatal or diseased hearts. We aimed to determine its existence, molecular nature in undiseased hearts and its potential arrhythmogenic implications under hyperactive conditions.Experimental Approach: Ventricular myocytes isolated from adult FVB mice were studied by using Ca2+ imaging and whole-cell perforated patch-clamp recording. In addition, lead II ECGs were recorded in isolated Langendorff-perfused mice hearts. Functional TRPC channel antibodies and inhibitors, and TRPC6 activator hyperforin were used.Key Results: In this study, we demonstrate the existence and contribution of SOCE in normal adult mouse cardiac myocytes. For an apparent SOCE activation, complete depletion of sarcoplasmic reticulum (SR) Ca2+ by employing both caffeine (10 mM) and thapsigargin (1 μM) or cyclopiazonic acid (10 μM) was required. Consistent with the notion that SOCE may be mediated by heteromultimeric TRPC channels, SOCEs observed from those myocytes were significantly reduced by the pretreatment with anti-TRPC1, 3, and 6 antibodies as well as by gadolinium, a non-selective TRPC channel blocker. In addition, we showed that SOCE may regulate spontaneous SR Ca2+ release, Ca2+ waves, and triggered activities which may manifest cardiac arrhythmias. Since the spontaneous depolarization in membrane potential preceded the elevation of intracellular Ca2+, an inward membrane current presumably via TRPC channels was considered as the predominant cause of cellular arrhythmias. The selective TRPC6 activator hyperforin (0.1–10 μM) significantly facilitated the SOCE, SOCE-mediated inward current, and calcium load in the ventricular myocytes. ECG recording further demonstrated the proarrhythmic effects of hyperforin in ex vivo mouse hearts.Conclusion and Implications: We suggest that SOCE, which is at least partially mediated by TRPC channels, exists in adult mouse ventricular myocytes. TRPC channels and SOCE mechanism may be involved in cardiac arrhythmogenesis via promotion of spontaneous Ca2+ waves and triggered activities under hyperactivated conditions.

Methylene Blue Counteracts H2S-Induced Cardiac Ion Channel Dysfunction and ATP Reduction

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H2S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca2+ homeostasis disrupted by H2S poisoning. In vivo, MB restores cardiac contractility severely depressed by sulfide and protects against arrhythmias, ranging from bundle branch block to ventricular tachycardia or fibrillation. To dissect the cellular mechanisms by which MB reduces arrhythmogenesis and improves bioenergetics in myocytes intoxicated with H2S, we evaluated the effects of H2S on resting membrane potential (Em), action potential (AP), Na+/Ca2+ exchange current (INaCa), depolarization-activated K+ currents and ATP levels in adult mouse cardiac myocytes and determined whether MB could counteract the toxic effects of H2S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H2S (100µM) significantly depolarized Em, reduced AP amplitude, prolonged AP duration at 90% repolarization (APD90), suppressed INaCa and depolarization-activated K+ currents, and reduced ATP levels in adult mouse cardiac myocytes. Treating cardiomyocytes with MB (20µg/ml) 3min after H2S exposure restored Em, APD90, INaCa, depolarization-activated K+ currents, and ATP levels toward normal. MB improved mitochondrial membrane potential (∆ψm) and oxygen consumption rate in myocytes in which Complex I was blocked by rotenone. We conclude that MB ameliorated H2S-induced cardiomyocyte toxicity at multiple levels: (1) reversing excitation-contraction coupling defects (Ca2+ homeostasis and L-type Ca2+ channels); (2) reducing risks of arrhythmias (Em, APD, INaCa and depolarization-activated K+ currents); and (3) improving cellular bioenergetics (ATP, ∆ψm).

Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

AimJunctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca2+ entry and intracellular Ca2+ release. The small‐conductance Ca2+‐activated K+ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.MethodsAssociation of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real‐time PCR. The expression of proteins was analysed by Western blotting. Ca2+‐activated K+ current (I K,Ca) in infected adult mouse cardiac myocytes was recorded using whole‐cell voltage‐clamp technique. The intracellular Ca2+ transient was measured using an IonOptix photometry system.ResultsWe showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N‐terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I K,Ca and reduced the amplitude of the Ca2+ transient in infected cardiomyocytes.ConclusionThe present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.

The 10 Most Read Articles Published in Circulation Research in 2016.

The 10 Most Read Articles Published in Circulation Research in 2016.

PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β-stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.

In This Issue

HomeCirculation ResearchVol. 119, No. 8In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published30 Sep 2016https://doi.org/10.1161/RES.0000000000000124Circulation Research. 2016;119:887Simplified Isolation of Adult Mouse Cardiac Myocytes (p 909)Download figureDownload PowerPointAckers-Johnson et al describe a convenient technique for isolating cardiomyocytes.For the last four decades, the method of choice for isolating adult mouse cardiomyocytes has been to excise the heart, mount it on perfusion apparatus (known as a Langendorff system), and pump dissociation buffers into the chambers under pressure—ensuring their penetration deep into the myocardium to dissociate the tissue into single cells. While this technique reproducibly yields myocytes, perfusion of the isolated heart requires significant expertise and specialized equipment, which are a barrier to many researchers. Ackers-Johnson and colleagues now report that they could attain a hydrodynamic pressure similar to that of the Langendorff apparatus by simply clamping the aorta and injecting buffer directly into the heart wall. In this method, they first flushed the heart with anticoagulant, then clamped the aorta, excised the heart, and bathed it in dissociation buffer while also injecting the buffer into the left ventricle. Once tissue disruption was apparent, gentle agitation separated the cells. The team found that the yield and quality of the isolated cardiomyocytes was similar to that obtained with the Langendorff system, with cells retaining their characteristic morphology, transcription profile, and function. This faster and simpler approach, which requires only standard surgical and laboratory equipment, should enable more researchers to readily obtain large quantities of viable primary cardiac myocytes for single-cell studies.Mesenchymal Stem Cells Stimulate Cardiac Stem Cells (p 921)Download figureDownload PowerPointHatzistergos et al investigate the stimulatory effects of mesenchymal stem cells on cardiac stem cells.Cardiac stem cells (CSCs), which express the cell surface marker c-kit, have been investigated for their capacity to replenish cardiomyocytes in damaged heart tissue. However, recent reports have suggested that the contribution of CSCs to endogenous cardiomyogenesis is relatively low. Other reports suggest that the cardiomyogenic potential of transplanted CSCs may be boosted in the heart by other cell types. For example, in a porcine model of chronic ischemic cardiomyopathy, mesenchymal stem cells (MSCs) have been reported to work synergistically with CSCs to improve cardiac function. The mechanism by which MSCs enhance CSCs is unclear, however. MSCs are known to secrete both stem cell factor (SCF), which interacts with c-kit, and stromal cell-derived factor (SDF), which interacts with cell surface protein CXCR4. Hatzistergos and colleagues now show that MSCs stimulate CSC proliferation via SCF-c-kit signaling, and affect CSC migration and differentiation via SDF-CXCR4 signaling. Although the team found that endogenous CSCs possess minimal cardiomyogenic potential, their results indicate that modulating SCF-c-kit and SDF1-CXCR4 signaling could maximize this potential. These findings strengthen the rationale for further investigations of cell-combination therapies for myocardial regeneration.Gut Microbiome and Cardiovascular Disease Risk (p 956)Download figureDownload PowerPointComposition of the gut microbiome reflects lifetime risk of cardiovascular disease, report Kelly et al.It is being increasingly appreciated that the collection and diversity of a person’s gut microbes (the microbiota) can influence their health. For example, particular microbiota compositions have been associated with obesity, diabetes, and other risk factors for cardiovascular disease (CVD). However, it is unclear whether gut microbiota composition is associated with a person’s overall CVD risk. To address this gap, Kelly and colleagues examined 112 participants (55 determined to be at high risk of CVD, and 57 at low risk) from the Bogalusa Heart Study (BHS)—a long-term epidemiological study of the lifetime development of atherosclerosis. The team took stool samples from the participants, isolated microbial DNA, and performed deep sequencing to determine which microbial species were present. They found that increased microbial diversity was consistently associated with lower CVD risk. They also identified specific genera of bacteria that were significantly associated with either high or low CVD risk. Although cause-and-effect relationships between microbiota composition and CVD could not be established, such a link, if validated, would suggest that microbiome-altering treatments could lessen CVD risk. Previous Back to top Next FiguresReferencesRelatedDetails September 30, 2016Vol 119, Issue 8 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000124 Originally publishedSeptember 30, 2016 PDF download Advertisement

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.

Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology.

Abstract 39: CD47 Regulates Aberrant Ca2+ Channel Current in Cardiac Myocytes

Background: Left ventricular (LV) heart failure (HF) affects over 5 million Americans with 50% of HF patients dying within 5 years of diagnosis. Therapeutics, while only relieving symptoms and have multiple side effects, have not completely resolved this process. HF is associated with cardiac myocyte cytosolic Ca2+ overload due to impaired sarcoplasmic reticulum (SR) and/or membrane Ca2+ handling. We recently found that a widely expressed cell surface receptor CD47 and its high affinity ligand, thrombospondin-1 (TSP1), are expressed on cardiac myocytes. We further showed that activated CD47 increases isolated neonatal cardiac myocyte cytosolic Ca2+ levels in part through SR Ca2+ channels. We postulated that TSP1-CD47 signaling may in part play a role in LV HF secondary to dysregulation of Ca2+, though the specific Ca2+ dynamics through which TSP1-CD47 signaling controls cardiac Ca2+ remains to be determined. Methods: Isolated hearts from mutated mice lacking CD47 (CD47-/-) and wild type (WT) mice with and without 4 weeks of transverse aortic constriction (TAC) were studied in ex vivo perfused heart Langendorff using dual optical mapping of Cai transients and action potential or voltage measurements. Isolated cardiac myocyte experiments were performed using whole cell voltage-clamp analysis. Results: In TAC-stressed WT murine hearts studied in the Landendorff system, tetracaine (an inhibitor of SR ryanodine receptor2) blunted arrhythmia induced by electrical stimulation. Isoproterenol-induced Ca2+ transient irregularities in hearts from WT mice were ameliorated in CD47-/- mice. Further, hearts from WT mice treated with peptide 7N3, a TSP1 mimetic, showed induced Ca2+ transient irregularities and elicited early after depolarization which was also attenuated with K2O1 (ryanodine receptor2 stabilizer) treatment. Voltage-clamp studies of isolated adult mouse cardiac myocytes showed that 7N3 peptide increased the L-type Ca2+ channel current (ICa,L) whereas the scrambled control peptide did not alter ICa,L. Conclusion: These data demonstrate that CD47 activation may be a trigger for aberrant myocyte Ca2+ handling typically associated with HF. Targeting of TSP1-CD47 signaling may open a new therapeutic avenue to be further studied in HF.

Abstract 339: DWORF: a Novel Cardiac Micropeptide That Enhances SERCA Activity and Cardiomyocyte Contractility

Background: Our lab has discovered a novel conserved micropeptide of 34 amino acids encoded by a muscle-specific RNA that was previously annotated as a putative long non-coding RNA (lncRNA). This micropeptide, which we named DWORF (DWarf Open Reading Frame), shares structural similarity with the known SERCA modulators phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN). Objective: To define the functional role of DWORF in the heart and elucidate its mechanism of action. Methods and Results: Immunofluorescence staining and imaging in isolated adult mouse cardiac myocytes indicates that DWORF specifically localizes to sarcoplasmic reticulum (SR) membranes where it co-localizes with SERCA. Co-IP experiments performed in COS or HEK cells co-transfected with DWORF and SERCA show that DWORF forms a stable complex with various SERCA isoforms. Further biochemical analysis indicates that DWORF binds to the same site on SERCA as PLN, and overexpression of DWORF is capable of competing with PLN for SERCA binding. Co-expression of GFP-tagged DWORF and PLN in the presence of SERCA followed by SERCA pull-down and GFP Western indicates that PLN and DWORF have similar affinities for SERCA binding. Transgenic mice with cardiac specific over-expression of DWORF show enhanced cellular contractility as evidenced by increased peak Ca2+ transient amplitude and faster cytosolic Ca2+ decay rates (reduced Tau values). Furthermore, Ca2+-dependent Ca2+-uptake assays performed in homogenates from DWORF transgenic and littermate control hearts show a statistically significant leftward shift of the Ca2+-dependence curve for SERCA indicating a higher affinity of SERCA for Ca2+. Intriguingly, DWORF expression is silenced in the heart in response to pathological calcineurin signaling, implicating this micropeptide in the cellular pathway for calcineurin action. Conclusions: We have identified a novel muscle-specific micropeptide, DWORF, which is expressed in the heart at high levels and has the capacity to bind SERCA and modulate its function to increase contractility in transgenic mice. Our findings provide exciting data in support of DWORF as a previously unrecognized endogenous SERCA activator that plays a role in cardiac contractility.

Interdependence of Parkin-Mediated Mitophagy and Mitochondrial Fission in Adult Mouse Hearts.

The role of Parkin in hearts is unclear. Germ-line Parkin knockout mice have normal hearts, but Parkin is protective in cardiac ischemia. Parkin-mediated mitophagy is reportedly either irrelevant, or a major factor, in the lethal cardiomyopathy evoked by cardiac myocyte-specific interruption of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. To understand the role of Parkin-mediated mitophagy in normal and mitochondrial fission-defective adult mouse hearts. Parkin mRNA and protein were present at low levels in normal mouse hearts, but were upregulated after cardiac myocyte-directed Drp1 gene deletion in adult mice. Alone, forced cardiac myocyte Parkin overexpression activated mitophagy without adverse effects. Likewise, cardiac myocyte-specific Parkin deletion evoked no adult cardiac phenotype, revealing no essential function for, and tolerance of, Parkin-mediated mitophagy in normal hearts. Concomitant conditional Parkin deletion with Drp1 ablation in adult mouse hearts prevented Parkin upregulation in mitochondria of fission-defective hearts, also increasing 6-week survival, improving ventricular ejection performance, mitigating adverse cardiac remodeling, and decreasing cardiac myocyte necrosis and replacement fibrosis. Underlying the Parkin knockout rescue was suppression of Drp1-induced hyper-mitophagy, assessed as ubiquitination of mitochondrial proteins and mitochondrial association of autophagosomal p62/sequestosome 1 (SQSTM1) and processed microtubule-associated protein 1 light chain 3 (LC3-II). Consequently, mitochondrial content of Drp1-deficient hearts was preserved. Parkin deletion did not alter characteristic mitochondrial enlargement of Drp1-deficient cardiac myocytes. Parkin is rare in normal hearts and dispensable for constitutive mitophagic quality control. Ablating Drp1 in adult mouse cardiac myocytes not only interrupts mitochondrial fission, but also markedly upregulates Parkin, thus provoking mitophagic mitochondrial depletion that contributes to the lethal cardiomyopathy.

Insulin Attenuates Cardiac Myocyte Contractility via NADPH Oxidase: Implications for Diabetic Cardiomyopathy

Insulin is known to modulate metabolic responses in cardiac myocytes (CM), yet the role of insulin in regulation of CM contractility is less well understood. Type 2 diabetes mellitus (T2D) leads to diabetic cardiomyopathy, yet little is known about the role of insulin in CM function. Here we explore the role of insulin on CM contractility in adult mouse CM. Insulin had no effect on basal CM contractility, but inhibited the increase in contractility seen after stimulation of ß‐adrenergic receptors (ß‐AR) by isoproterenol (ISO; 40+/‐5% inhibition, p<0.01, n=115‐124 cells). The attenuation of ISO‐stimulated contractility by insulin was blocked by the NADPH oxidase inhibitor apocynin, implicating reactive oxygen species (ROS) in cardiac insulin responses. We found that insulin inhibited ß‐AR stimulation of CM Ca2+ transients (35+/‐4% inhibition, p<0.01, n=39‐41 cells); again, insulin inhibition of this ß‐AR response was blocked by apocynin. We analyzed CM contractile responses in CM isolated from mice fed a high‐fat diet, a model of T2D. We found that the insulin‐promoted decrease in contractility was blocked in CM isolated from diabetic mice. The phosphorylation of insulin‐dependent signaling proteins (insulin receptor, IRS‐1, Akt) was effectively abrogated in diabetic mice, consistent with myocyte insulin resistance. These data establish a central role for NADPH oxidase in insulin‐dependent physiological responses in CM, and show that these responses are deranged in mice with T2D. These observations suggest that alterations in ROS‐ and insulin‐dependent pathways play a role in the pathophysiology of diabetic cardiomyopathy. Research support: NIH and AHA.

A Central Role for H2O2 in Insulin Signal Transduction in Cardiac Myocytes

Molecular mechanisms of insulin action have been extensively characterized in the classic insulin target tissues: fat, liver, and skeletal muscle. But in cardiac myocytes (CM), understanding of insulin‐modulated signaling pathways has lagged far behind. Early studies of insulin action implicated a physiological role for H2O2, but the relationships between insulin and H2O2 in CM signal transduction are almost entirely unexplored. In these studies, we identified a role for H2O2 in insulin signaling pathways in adult mouse CM. Insulin promoted the rapid (maximal at 5 min) robust (10‐ to 18‐fold) and reversible phosphorylation of insulin receptorβTyr1146/1150; IRS‐1 Tyr895; and Akt Ser473/308; these responses all had an EC50 of 1 nM insulin (n=3‐7). Insulin‐dependent phosphorylation of IRS‐1 Ser636/639, mTOR Ser2448, p70S6 Thr389/421, and S6 Ser235/Ser236 showed a slower time course (maximal at 30 min), again with an EC50 of 1 nM insulin and with robust phosphorylation responses (6‐ to 40‐fold increases from baseline; n=3‐7). All of these phosphorylation responses were blocked by the NADPH oxidase inhibitors apocynin and VAS2870, implicating NADPH oxidase‐derived reactive oxygen species in modulating insulin‐dependent phosphorylation responses. Importantly, the cell‐permeant derivatized enzyme PEG‐catalase (which degrades H2O2) attenuated all these insulin‐dependent phosphorylation responses. Our findings implicate NADPH oxidase‐generated H2O2 as a critical determinant of insulin‐dependent signaling responses in CM, and may have important implications for the abnormalities in cardiac function seen in disease states associated with oxidative stress. Research support: NIH and AHA.

EHD2 Stabilizes the Surface Expression of Cardiac KATP Channels and Protects Against Ischemic Injury

Opening of ATP‐sensitive potassium (KATP) channels mediate ischemic preconditioning (IPC), which is an endogenous protection mechanism against ischemic injury. We found that internalization of KATP channels during ischemia is prevented by IPC. We investigated the role of EHD2 (identified in our proteomic assays as a KATP channel interacting protein) in KATP channel internalization. A dominant‐negative EHD2‐G65R caused Kir6.2/SUR2A KATP channels to redistribute in transfected COS‐7 cells. Both the dominant negative Rab11‐S25N and EHD2‐G65R led to accumulation of Kir6.2 in the endocytic recycling complex (ERC), suggesting that EHD2 controls exit from the ERC to the plasma membrane. Using biotinylation assays, we found that EHD2 decreased (whereas EHD2‐G65R accelerated) the rate of KATP channel endocytosis. Surface biotinylation experiments showed that EHD2 stabilized and EHD2‐G65R decreased the surface density of Kir6.2/SUR2A channels in HEK‐293 cells. Consistently, patch clamping demonstrated that EHD2 increases, whereas EHD2‐G65R decreases, KATP channel current density without affecting KATP channel properties. Similar data were obtained with isolated adult mouse cardiac myocytes infected with Ad‐mCherry‐EHD2 or Ad‐mCherry‐EHD2‐G65R. We employed a cellular model of pharmacological preconditioning, in which myocytes are pretreated with phenylephrine prior to ischemic pelleting. Adult mouse cardiomyocytes infected with Ad‐mCherry‐EHD2 resisted ischemic injury, whereas cell death was more prominent in Ad‐mCherry‐EHD2‐G65R. Our data demonstrate that EHD2 positively regulates KATP channel surface expression and participates in the protection afforded by preconditioning.

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes.

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.

Cell Signaling Associated with Na+/K+-ATPase: Activation of Phosphatidylinositide 3-Kinase IA/Akt by Ouabain Is Independent of Src

Exposure of intact cells to selective inhibitors of Na+/K+-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these pathways is the binding of ouabain to a preexisting complex of Src with Na+/K+-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src++ cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src++ cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na+/K+-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src++ cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na+/K+-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na+/K+-ATPase and (b) a more likely, but not established, mechanism of linkage of Na+/K+-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na+/K+-ATPase with the SH3 domain of the p85 subunit of PI3K1A.