Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture.

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor-mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine-induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada -/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Thoracic aortic disease in tuberous sclerosis complex: molecular pathogenesis and potential therapies in Tsc2+/- mice.

Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^

Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^

Scaffolding of adenylyl cyclase by A Kinase Anchoring Proteins

Adenylyl cyclase (AC) converts ATP into cAMP, which activates protein kinase A (PKA). Activation of PKA leads to the phosphorylation of specific substrates. The mechanism of specificity of PKA phosphorylation baffled researchers for many years. The discovery of A Kinase Anchoring Proteins (AKAPs) has helped to unravel this mystery. AKAPs function to target PKA to specific regions within the cell. They also anchor other enzymes, receptors, or channels leading to tightly regulated signaling modules. Several studies have suggested an important role for activated PKA in these complexes, including the AKAPs yotiao and muscle AKAP (mAKAP). Yotiao, a plasma membrane AKAP, anchors PP1, NMDA receptors, IP3 receptors, and heart potassium channel subunit KCNQI. PKA phosphorylation of NMDA receptors as well as KCNQI leads to increased channel activity. Patients with mutations in KCNQI or yotiao that cause loss of targeting of KCNQI develop long QT syndrome, which can be fatal. mAKAP anchors several CAMP/PKA-regulated pathways to the nuclear envelope in cardiac myocytes. The necessity of activated PKA in these complexes led to the hypothesis that AC is also anchored. The results indicate that AC does associate with yotiao in brain and heart, specifically with AC types I-III, and IX. Co-expression of AC II or III with yotiao leads to inhibition of each isoform's activity. Binding assays revealed that yotiao binds to the N-terminus of AC II and that this region can reverse the inhibition of AC II, but not AC III, indicating unique binding sites on yotiao. AC II binds directly to as 808-957 of yotiao. Y808-957 acts as a dominant negative as the addition of it to rat brain membranes results in a ∼40% increase in AC activity. Additionally, AC was also found to associate with mAKAP in heart, specifically with AC types II and V. The binding site of AC was mapped to 275-340 of mAKAP, while mAKAP binds to the soluble domains of AC V as a complex. These results indicate that interactions between AC and AKAPs are specific and that AC plays an important role in AKAP-targeted signaling. ^

The role of transforming growth factor beta signaling in cardiac hypertrophy and fibrosis in heart failure

Objective. To determine whether transforming growth factor beta (TGF-β) receptor blockade using an oral antagonist has an effect on cardiac myocyte size in the hearts of transgenic mice with a heart failure phenotype. ^ Methods. In this pilot experimental study, cardiac tissue sections from the hearts of transgenic mice overexpressing tumor necrosis factor (MHCsTNF mice) having a phenotype of heart failure and wild-type mice, treated with an orally available TGF-β receptor antagonist were stained with wheat germ agglutinin to delineate the myocyte cell membrane and imaged using fluorescence microscopy. Using MetaVue software, the cardiac myocyte circumference was traced and the cross sectional area (CSA) of individual myocytes were measured. Measurements were repeated at the epicardial, mid-myocardial and endocardial levels to ensure adequate sampling and to minimize the effect of regional variations in myocyte size. ANOVA testing with post-hoc pairwise comparisons was done to assess any difference between the drug-treated and diluent-treated groups. ^ Results. There were no statistically significant differences in the average myocyte CSA measured at the epicardial, mid-myocardial or endocardial levels between diluent treated littermate control mice, drug treated normal mice, diluent-treated transgenic mice and drug-treated transgenic mice. There was no difference between the average pan-myocardial cross sectional area between any of the four groups mentioned above. ^ Conclusions. TGF-β receptor blockade using oral TGF-β receptor antagonist does not alter myocyte size in MHCsTNF mice that have a phenotype of heart failure. ^

Metabolic Regulation of mTOR Activation and Endoplasmic Reticulum Stress in the Heart

When subjected to increased workload, the heart responds metabolically by increasing its reliance on glucose and structurally by increasing the size of myocytes. Whether changes in metabolism regulate the structural remodeling process is unknown. A likely candidate for a link between metabolism and growth in the heart is the mammalian target of rapamycin (mTOR), which couples energy and nutrient metabolism to cell growth. Recently, sustained mTOR activation has also been implicated in the development of endoplasmic reticulum (ER) stress. We explored possible mechanisms by which acute metabolic changes in the hemodynamically stressed heart regulate mTOR activation, ER stress and cardiac function in the ex vivo isolated working rat heart. Doubling the heart’s workload acutely increased rates of glucose uptake beyond rates of glucose oxidation. The concomitant increase in glucose 6-phosphate (G6P) was associated with mTOR activation, endoplasmic reticulum (ER) stress and impaired contractile function. Both rapamycin and metformin restored glycolytic homeostasis, relieved ER stress and rescued contractile function. G6P and ER stress were also downregulated with mechanical unloading of failing human hearts. Taken together, the data support the hypothesis that metabolic remodeling precedes, triggers, and sustains structural remodeling of the heart and implicate a critical role for G6P in load-induced contractile dysfunction, mTOR activation and ER stress. In general terms, the intermediary metabolism of energy providing substrates provides signals for the onset and progression of hypertrophy and heart failure.

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^