Cardiac Myocyte Function Research Articles

Carbon nanotubes enhance intercalated disc assembly in cardiac myocytes via the β1-integrin-mediated signaling pathway

Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the β1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.

Nuclear compartmentalization of α1-adrenergic receptor signaling in adult cardiac myocytes.

:Although convention dictates that G protein-coupled receptors localize to and signal at the plasma membrane, accumulating evidence suggests that G protein-coupled receptors localize to and signal at intracellular membranes, most notably the nucleus. In fact, there is now significant evidence indicating that endogenous alpha-1 adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. Cumulatively, the data suggest that α1-ARs localize to the inner nuclear membrane, activate intranuclear signaling, and regulate physiologic function in adult cardiac myocytes. Although α1-ARs signal through Gαq, unlike other Gq-coupled receptors, α1-ARs mediate important cardioprotective functions including adaptive/physiologic hypertrophy, protection from cell death (survival signaling), positive inotropy, and preconditioning. Also unlike other Gq-coupled receptors, most, if not all, functional α1-ARs localize to the nuclei in adult cardiac myocytes, as opposed to the sarcolemma. Together, α1-AR nuclear localization and cardioprotection might suggest a novel model for compartmentalization of Gq-coupled receptor signaling in which nuclear Gq-coupled receptor signaling is cardioprotective.

Cardiac myocyte dysfunction induced by streptolysin O is membrane pore and calcium dependent.

Septic cardiomyopathy is a severe complication among some patients who develop group A streptococcal toxic shock syndrome. Despite the importance of cardiac dysfunction in determining prognosis, very little is known about mechanisms that reduce cardiac output in association with streptococcal infection. Here, we investigated the effects of streptococcal extracellular toxins on mechanical contractility of electrically paced primary murine cardiomyocytes. Our data demonstrate that streptolysin O (SLO) is the major streptococcal toxin responsible for cardiomyocyte contractile dysfunction. Streptolysin O dose-dependently affected cardiac myocyte function in discrete stages. Exposure to SLO caused a failure of cardiac cells to respond to electrical pacing, followed by spontaneous dysregulated contractions and augmented strength of contraction. Central to these SLO-mediated effects is a marked influx of calcium into the cytosol through SLO-mediated pores in the cytoplasmic membrane. Such calcium mobilization in response to SLO correlated temporally with hypercontractility and unpaced contractions. During continued exposure to SLO, cardiomyocytes exhibited periods of reversion to normal electrical pacing suggestive of membrane lesion repair and restoration of calcium handling. Together, these observations are consistent with the clinical observation that septic cardiomyopathy is a reversible condition in patients who survive streptococcal toxic shock syndrome. These data provide strong evidence that streptococcal exotoxins, specifically SLO, can directly impact cardiac mechanical function.

Species-dependent adaptation of the cardiac Na+/K+ pump kinetics to the intracellular Na+ concentration.

The Na(+)/K(+) ATPase (NKA) plays a critical role in maintaining ionic homeostasis and dynamic function in cardiac myocytes, within both the in vivo cell and in silico models. Physiological conditions differ significantly between mammalian species. However, most existing formulations of NKA used to simulate cardiac function in computational models are derived from a broad range of experimental sources spanning many animal species. The resultant inability of these models to discern species-specific features is a significant obstacle to achieving a detailed quantitative and comparative understanding of physiological behaviour in different biological contexts. Here we present a framework for characterising the steady-state NKA current using a biophysical mechanistic model specifically designed to provide a mechanistic explanation of the NKA flux supported by self-consistent species-specific data. We thus compared NKA kinetics specific to guinea- pig and rat ventricular myocytes. We observe that the apparent binding affinity for sodium in the rat is significantly lower, whereas the overall pump cycle rate is doubled, in comparison to the guinea pig. This sensitivity of NKA to its regulatory substrates compensates for the differences in Na(+) concentrations between the cell types. NKA is thereby maintained within its dynamic range over a wide range of pacing frequencies in these two species, despite significant disparities in sodium concentration. Hence, by replacing a conventional generic NKA model with our rat-specific NKA formula into a whole-cell simulation, we have, for the first time, been able to accurately reproduce the action potential duration and the steady-state sodium concentration as functions of pacing frequency.

Circulation Research “In This Issue” Anthology

<i>Circulation Research</i> “In This Issue” Anthology

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca2+ sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.

Work and Tension: New Evidence that Adherent Cells of the Same Area Do the Same Work Independent of Stiffness and Focal Adhesions

Work and Tension: New Evidence that Adherent Cells of the Same Area Do the Same Work Independent of Stiffness and Focal Adhesions

Abstract 177: Promotion Of Nitroso-redox Balance By Beta 3 Adrenoceptor Agonism: Therapeutic Implications For Cardiovascular Complications Of Diabetes

Rationale: Disrupted balance between NO and O2.- is central in pathobiology of diabetes-induced cardiomyopathy and vascular dysfunction. We examined if stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re-establish NO/O2.- balance, relieve oxidative inhibition of key caveolar proteins and protect against diabetes-induced cardiovascular dysfunction. Methods/Results: A hyperglycemic, hyperinsulinemic state was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 (12 μg/kg/h). Diabetes induced NADPH oxidase-dependent glutathionylation (GSS-) of the caveolar proteins Na+-K+ pump’s β1 subunit and eNOS in cardiac myocytes and aorta, an oxidative modification that inhibits the pump and uncouples eNOS. Consistent with this, diabetes was associated with reduced electrogenic Na+-K+ pump current in voltage-clamped cardiac myocytes and impaired endothelium-dependent vasorelaxation. Selective β3 AR agonist CL316243 (CL, 40 μg/kg/h) restored NO levels analysed by spin-trapping of NO-Fe(DETC)2 complexes; decreased diabetes-induced elevation in O2.- measured by HPLC analysis of dihydroethidium oxidation products, improved endothelium-dependent vasorelaxation, and restored the Na+-K+ pump function in cardiac myocytes. These effects were mediated by CL abolishing diabetes-induced increase in eNOS-GSS and β1-GSS through a decrease in forward reaction rate for glutathionylation by suppressing diabetes-induced NADPH oxidase activation, which was further amplified by promotion of de-glutathionylation via enhancement in association of glutaredoxine-1, the enzyme catalysing de-glutathionylation, with eNOS and Na+-K+ pump. Conclusion: β3 AR activation re-established nitroso-redox balance and relieved oxidative inhibition of key caveolar proteins in diabetes. β3 AR agonists are promising in treatment of diabetes-induced cardiovascular complications.

Abstract 151: MCL-1 Promotes Mitochondrial Fusion and Survival in Cardiac Myocytes

Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is highly expressed in myocardium, but little is known about its function in myocytes. Recently, we reported that MCL-1 is essential for myocardial homeostasis and autophagy. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Despite extensive mitochondrial damage, MCL-1 deficient hearts failed to activate mitochondrial autophagy. Parkin, an E3 ubiquitin ligase, normally translocates to damaged mitochondria to promote mitochondrial autophagy, but loss of MCL-1 resulted in cytosolic accumulation of Parkin. However, we found no evidence that MCL-1 functions as a mitochondrial Parkin receptor or substrate. Instead, loss of MCL-1 reduced mitochondrial accumulation of PINK1, which is involved in Parkin recruitment. Additionally, we identified mitochondrial outer membrane (OM) and matrix isoforms of MCL-1 in mouse hearts and found that the two forms respond differently to ischemic injury. Four hours after myocardial infarction, MCL-1 OM levels were reduced by 40% in border zone tissue. After 24 hours, MCL-1 OM levels returned to baseline. Meanwhile, MCL-1 Matrix levels were preserved at four hours, and increased significantly compared to control 24 hours after infarction. These changes correlated with increased expression of HSP70, a chaperone protein that stabilizes MCL-1 and participates in import of mitochondrial proteins. Overexpression of MCL-1 Matrix promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission. While 94.1% (1269 of 1349) of control cells exhibited fragmented mitochondria after two hours of FCCP treatment, mitochondria were only fragmented in 41.3% (391 of 947) of cells overexpressing MCL-1 Matrix . These data suggest MCL-1 isoforms play different roles in the cellular stress response. MCL-1 OM protects against apoptosis, whereas MCL-1 Matrix protects mitochondria by promoting fusion. In addition, upregulation of HSP70 may preserve mitochondrial function and cell viability in damaged cardiac myocytes by increasing the stability and mitochondrial import of MCL-1.

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

CRTC2 function in cardiac myocytes (699.5)

CRTC2 (cAMP‐regulated transcription co‐activator 2) is a transcription co‐factor which integrates signaling pathways activated by β‐adrenergic receptor (βAR) and Ca2+ in metabolism. In this study, we tested the hypothesis that increased CRTC2 expression in cardiac myocytes would be equivalent to sustained βAR and Ca2+ signaling, and could have a deleterious effect on the function of cardiac myocytes. To our surprise, decreased CRTC2 protein content was found in pressure overloaded hearts. CRTC2 expression in isolated cardiac myocytes was associated with blunted αAR‐stimulated hypertrophic growth without affecting cell proliferation. Expression of CRTC2 also blocked re‐activation of the fetal gene program, and altered expression of critical regulators of cardiac hypertrophy. Furthermore, CRTC2 affected expression of factors which are pivotal for Ca2+ handling and apoptosis. The effects of nuclear‐targeted CRTC2 [ntCRTC2; i.e. CRTC2(S171A)] was more profound than CRTC2 itself. Using the newly generated ntCRTC2 transgenic mice, we found that ntCRTC2 expression attenuated LV hypertrophy and dysfunction in vivo in pressure overload. These results demonstrate the functional importance of CRTC2 in cardiac hypertrophy and function.

Nuclear Localization of α1A‐Adrenergic Receptors Is Required for Signaling in Cardiac Myocytes: An “Inside‐Out” α1‐AR Signaling Pathway

BackgroundRecent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes.Methods and ResultsBy measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI.ConclusionsTogether, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.

Localization and Role of Transient Receptor Potential Cation Channels in Rabbit Ventricular Myocytes

Several members of the transient receptor potential cation (TRPC) channel family are stretch activated. They are thought to play a critical role in mechano-electrical coupling in cardiac myocytes and in cardiac hypertrophic remodeling. However, studies on their subcellular localization in cardiomyocytes are conflicting, and their functions in cardiac myocytes are barely understood. In this study, we investigated the spatial distribution of TRPC channels in isolated ventricular myocytes from adult rabbit using immunolabeling and three-dimensional confocal microscopy. Colocalization of TRPC with (i) sarcolemma labeled with wheat germ agglutinin (WGA), (ii) sarcoplasmic reticulum (SR) labeled for SR calcium ATPase (SERCA2), and (iii) cytoskeletal proteins including sarcomeric α-actinin, desmin, vinculin and β-tubulin, was assessed by a quantitative approach. Furthermore, we measured stretch-activated current with whole-cell voltage clamping. Our results from confocal imaging revealed a transverse striated distribution of TRPC1 and TRPC6 channels. We found colocalization of TRPC1 and TRPC6 with sarcomeric α-actinin, SERCA2A and desmin, but not with WGA, vinculin and β-tubulin. Additionally, no stretch-activated current was detected when stretching isolated cardiomyocytes to approximately 10% of their resting length. Taken together, our data indicate that TRPC1 and TRPC6 are not located in the sarcolemma, but in the membrane of the SR adjacent to the Z-disks in rabbit ventricular myocytes. We suggest that a potential role of TRPC1 and TRPC6 channels is in stretch-dependent calcium leak from the SR and/or counter current balancing calcium release from the SR through ryanodine receptors.

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment.

Mechanosensing and Regulation of Cardiac Function.

The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. However, mechanical overload is a pathogenesis or comorbidity existing in a variety of heart diseases, such as hypertension, aortic regurgitation and myocardial infarction. Physical stimuli sensed by cells are transmitted through intracellular signal transduction pathways resulting in altered physiological responses or pathological conditions. Emerging evidence from experimental studies indicate that β1-integrin and the angiotensin II type I (AT1) receptor play critical roles as mechanosensors in the regulation of heart contraction, growth and leading to heart failure. Integrin link the extracellular matrix and the intracellular cytoskeleton to initiate the mechanical signalling, whereas, the AT1 receptor could be activated by mechanical stress through an angiotensin-II-independent mechanism. Recent studies show that both Integrin and AT1 receptor and their downstream signalling factors including MAPKs, AKT, FAK, ILK and GTPase regulate heart function in cardiac myocytes. In this review we describe the role of mechanical sensors residing within the plasma membrane, mechanical sensor induced downstream signalling factors and its potential roles in cardiac contraction and growth.

Recent Advances in Cardiac Myocyte Biology and Function

Cardiac myocyte dysfunction is the underlying cause of changes in cardiac contractility associated with several pathological conditions. Hence, the physiology of cardiac myocytes has been the subject of intense investigation and much attention has been paid to important questions such as the following: what maintains and regulates the structural and functional integrity of cardiac myocytes?; how do myocytes respond to stress?; what are the molecular changes that contribute to myocytes dysfunction in cardiac disease?; how can pathological changes in myocytes be prevented or attenuated?; what determines myocyte life and death?; and can adult myocytes be renewed or replenished? Recent advancements in the field continue to address these important issues.1–18 Cardiac myocyte hypertrophy has been an area of intense interest for a long time,19 because it is well-known that maladaptive cardiac myocyte hypertrophy and accompanying cardiac remodeling often lead to heart failure.20,21 Recent studies have uncovered new mechanisms of myocyte hypertrophy. For instance, Matsushima et al22 showed that increased nuclear oxidative stress caused by nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) leads to oxidation and nuclear export of histone deacetylase 4 (a class II histone deacetylase), which is a repressor of transcription factors involved in hypertrophic response (eg, nuclear factor of activated T-cells); this suggest that inhibition of Nox4 can be another avenue for dampening maladaptive cardiac hypertrophy. G-protein–coupled receptor kinase-5 (GRK5) is another important regulator/kinase of a class II histone deacetylase 5 and its contribution to cardiac hypertrophy had been implicated previously.23 Recently, Gold et al24 have used both global and cardiac-specific Grk5 knockout mice to test the requirement of this protein for pathological hypertrophy. In response to pressure overload, Grk5 knockout mice showed attenuated hypertrophy, remodeling, and hypertrophy-related gene expression, clearly demonstrating the essential role of this protein in …

Analysis of various physiological salines for heart rate, CNS function, and synaptic transmission at neuromuscular junctions in Drosophila melanogaster larvae

Drosophila serves as a playground for examining the effects of genetic mutations on development, physiological function and behavior. Many physiological measures that address the effects of mutations require semi-intact or cultured preparations. To obtain consistent physiological recordings, cellular function needs to remain viable. Numerous physiological salines have been developed for fly preparations, with emphasis on nervous system viability. The commonly used saline drifts in pH and will cause an alteration in the heart rate. We identify a saline that maintains a stable pH and physiological function in the larval heart, skeletal neuromuscular junction, and ventral nerve cord preparations. Using these common assays, we screened various pH buffers of differing concentrations to identify optimum conditions. Buffers at 25mM produce a stable heart rate with minimal variation in pH. Excitatory junction potentials evoked directly on larval muscles or through sensory-CNS-motor circuits were unaffected by at buffers at 25mM. The salines examined did not impede the modulatory effect of serotonin on heart rate or neural activity. Together, our results indicate that the higher buffer concentrations needed to stabilize pH in HL3 hemolymph-like saline do not interfere with the acute function of neurons or cardiac myocytes.

P.11.5 PMO-mediated dystrophin exon 23 skipping restores mitochondrial function in the mdx mouse heart

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the L-type Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phosphorodiamidate morpholino oligomer, designed to induce skipping of dystrophin exon 23, “restored” the increase in mitochondrial membrane potential in mdx cardiomyocytes after activation of the L-type Ca2+ channel with the dihydropyridine receptor agonist BayK (−). These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse heart.

CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes

BackgroundCIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.ResultsWe now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [3H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.ConclusionsThese results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

Abstract 093: Adrenergic Stimulation Induces Mitochondrial Fragmentation and Cell Injury through PKD1-dependent Phosphorylation of DLP1 in H9c2 Cardiac Myoblasts.

Introduction: Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiac myocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed in various pathophysiological states of human heart such as heart failure, where the catecholamine level is elevated. However, it is still unclear what kinds of cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Hypothesis: Adrenergic signaling induces cardiac mitochondrial morphology changes and mitochondrial dysfunction, which simultaneously contribute to cardiac injury. Methods: H9c2 cardiac myoblasts were stimulated by α 1 -adrenoceptor (α 1 -AR) agonist phenylephrine and mitochondrial morphology was monitored by confocal microscopy. Translocation and phosphorylation of a mitochondrial fission protein, dynamin-like protein 1 (DLP1) was observed from whole cell lysates, cytosolic proteins and mitochondrial proteins by western blotting. Results: We found that persistent α 1 -AR stimulation induced mitochondrial fragmentation, followed by an increase in the production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria to the cytosol in H9c2 cardiac myoblasts. These effects were abolished by the treatment of α 1 -AR antagonist, prazosin. Further, mitochondrial fragmentation by α 1 -AR stimulation was inhibited by expression of the dominant-negative fission mutant DLP1-K38A, suggesting that the mitochondrial fission is required for mitochondrial fragmentation observed in α 1 -AR stimulation. We also found that DLP1 was translocated from cytosol to mitochondria under α 1 -AR stimulation. In addition, activation of protein kinase D1 (PKD1), a protein kinase downstream of α 1 -AR signaling, led to the phosphorylation of DLP1 at serine 637 which lies within a putative PKD phosphorylation consensus motif. Conclusion: α 1 -AR signaling induces mitochondrial fragmentation and cell injury, possibly through PKD1-dependent phosphorylation of DLP1.