Primary Cultures Of Cardiac Myocytes Research Articles

NF-κB activation is cell type-specific in the heart

Viral myocarditis is common and can progress to cardiac failure. Cardiac cell pro-inflammatory responses are critical for viral clearance, however sustained inflammatory responses contribute to cardiac damage. The transcription factor NF-κB regulates expression of many pro-inflammatory cytokines, but basal and induced activation of NF-κB in different cardiac cell types have not been compared. Here, we used primary cultures of cardiac myocytes and cardiac fibroblasts to identify cardiac cell type-specific events. We show that while viral infection readily stimulates activation of NF-κB in cardiac fibroblasts, cardiac myocytes are largely recalcitrant to activation of NF-κB. Moreover, we show that cardiac myocyte subpopulations differ in their NF-κB subcellular localization and identify the cis-Golgi as a cardiac myocyte-specific host compartment. Together, results indicate that NF-κB-dependent signaling in the heart is cardiac cell type-specific, likely reflecting mechanisms that have evolved to balance responses that can be either protective or damaging to the heart.

Epigallocatechin-3-gallate and zinc provide anti-apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells.

It has previously been demonstrated that phospha-tidylinositol-3-kinase (PI3K)/Akt and cleaved caspase-3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin-3-gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription-1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3-h hypoxia, followed by 1-h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti-apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R-induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice.

Involvement of cholesterol efflux pathway in the control of cardiomyocytes cholesterol homeostasis

Although cholesterol-rich microdomains are highly involved in the functions of cardiomyocytes, the cholesterol homeostasis is largely unknown in these cells. We developed experimental procedures to assess cholesterol synthesis, cholesterol masses and cholesterol efflux from primary cultures of cardiac myocytes obtained from 2 to 4days old Wistar rats. We first observed that cardiomyocytes poorly internalized exogenously supplied native or modified LDL and that free cholesterol (FC) efflux to free apolipoprotein AI (apo AI) and to HDL was mediated by ATP binding cassette transporter A1 (ABCA1) and likely by ATP binding cassette transporter G1 (ABCG1), respectively, which are both upregulated by liver X receptor/retinoid X receptor (LXR/RXR) activation. We then investigated the consequences of cholesterol synthesis inhibition on cholesterol homeostasis using an HMGCoA reductase inhibitor (pravastatin, 90% effective concentration (EC90): 0.11mM, 18h). We observed no impact of cholesterol synthesis inhibition on the FC or cholesteryl ester (CE) masses. Consistently with no FC mass changes, pravastatin treatment had no notable impact on LDL receptors mRNA expression or on the capacity of cardiomyocytes to uptake radiolabeled LDL. Conversely, pravastatin treatment induced a significant decrease of cholesterol efflux to both apo AI and HDL whereas the passive aqueous diffusion remained unchanged. The cholesterol efflux pathway reductions induced by cholesterol synthesis inhibition were not caused by a reduction of ABC transporter expression (mRNA or protein). These results show that cardiac myocytes down-regulate active cholesterol efflux processes when endogenous cholesterol synthesis is inhibited, allowing them to preserve cholesterol homeostasis.

Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as--log(10) [free Zn(2+)]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three‐dimensional polymeric microstructures

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.

Western array analysis of cell cycle protein changes during the hyperplastic to hypertrophic transition in heart development

Cardiac myocytes proliferate most rapidly during the hyperplastic phase of heart development; however, the level of cell cycle activity is drastically down regulated after birth. Further growth of the heart is achieved by hypertrophic growth of cardiac myocytes. The mechanism that controls the switch from hyperplastic proliferation to hypertrophic growth in cardiac myocytes is unknown. Understanding this fundamental mechanism of cardiac myocyte biology would be most beneficial for studies directed towards myocardial regeneration. In this study, we identified changes in the expression of proteins involved in cell cycle regulation during the hyperplastic to hypertrophic transition of cardiac myocytes. Using a high-throughput immunoblotting technique, we examined 200+ proteins in primary cultures of cardiac myocytes at different developmental time points to determine the important regulators of this transition. In addition, we also analyzed samples from an immortalized cardiac myocyte cell line to compare expression levels of cell cycle regulatory proteins to our primary cultures. Our findings by this uncovered proteomic screen identified several potential key regulatory proteins and provide insight into the important components of cardiac myocyte cell cycle regulation.

MicroRNA miR-133 Represses HERG K+ Channel Expression Contributing to QT Prolongation in Diabetic Hearts

We have previously found that the ether-a-go-go related gene (ERG), a long QT syndrome gene encoding a key K(+) channel (I(Kr)) in cardiac cells, is severely depressed in its expression at the protein level but not at the mRNA level in diabetic subjects. The mechanisms underlying the disparate alterations of ERG protein and mRNA, however, remained unknown. We report here a remarkable overexpression of miR-133 in hearts from a rabbit model of diabetes, and in parallel the expression of serum response factor (SRF), which is known to be a transactivator of miR-133, was also robustly increased. Delivery of exogenous miR-133 into the rabbit myocytes and cell lines produced post-transcriptional repression of ERG, down-regulating ERG protein level without altering its transcript level and caused substantial depression of I(Kr), an effect abrogated by the miR-133 antisense inhibitor. Functional inhibition or gene silencing of SRF down-regulated miR-133 expression and increased I(Kr) density. Repression of ERG by miR-133 likely underlies the differential changes of ERG protein and transcript thereby depression of I(Kr), and contributes to repolarization slowing thereby QT prolongation and the associated arrhythmias, in diabetic hearts. Our study provided the first evidence for the pathological role of miR-133 in adult hearts and thus expanded our understanding of the cellular function and pathophysiological roles of miRNAs.

Tyrosine Phosphorylation of Kir3.1 in Spinal Cord Is Induced by Acute Inflammation, Chronic Neuropathic Pain, and Behavioral Stress

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.

Basal and Reovirus-Induced Beta Interferon (IFN-β) and IFN-β-Stimulated Gene Expression Are Cell Type Specific in the Cardiac Protective Response

Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-beta) and IFN-beta-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-beta response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-beta response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-beta, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-beta), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-beta, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-beta in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-beta resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-beta response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-beta, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-beta and ISG expression in myocytes may play an important protective role.

Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells

It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific.

Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in regulating genome stability, cell cycle progression, and cell survival. However, overactivation of PARP has been shown to contribute to cell death and organ failure in various stress-related disease conditions. In this study, we examined the role of PARP in the development and progression of cardiac hypertrophy. We measured the expression of PARP in mouse hearts with physiological (swimming exercise) and pathological (aortic banding) cardiac hypertrophy as well as in human heart samples taken at the time of transplantation. PARP levels were elevated both in swimming and banded mice hearts and demonstrated a linear positive correlation with the degree of cardiac hypertrophy. A dramatic increase (4-fold) of PARP occurred in 6-wk banded mice, accompanied by apparent signs of ventricular dilation and myocyte cell death. PARP levels were also elevated (2- to 3-fold) in human hearts with end-stage heart failure compared with controls. However, we found no evidence of caspase-mediated PARP cleavage in either mouse or human failing hearts. Overexpression of PARP in primary cultures of cardiac myocytes led to suppression of gene expression and robust myocyte cell death. Furthermore, data obtained from the analysis of PARP knockout mice revealed that these hearts produce an attenuated hypertrophic response to aortic banding compared with controls. Together, these results demonstrate a role for PARP in the onset and progression of cardiac hypertrophy and suggest that some events related to cardiac hypertrophy growth and progression to heart failure are mediated by a PARP-dependent mechanism.

Ras, Akt, and mechanotransduction in the cardiac myocyte.

The Ras subfamily of 21-kDa ("small") guanine nucleotide binding proteins [which includes Ha-Ras, Ki(A)-Ras, Ki(B)-Ras, and N-Ras] is universally important in regulating intracellular signaling events in mammalian cells and controls their growth, proliferation, senescence, differentiation, and survival. These Ras isoforms act as membrane-associated biological switches that transduce signals from transmembrane receptors, thus potentially activating a variety of downstream signaling proteins. These include ultimately two Ser/Thr protein kinase families, the extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt (or protein kinase B). Activation of ERK1/2 has been associated with cardiac myocyte hypertrophy (ie, increased cell size and myofibrillogenesis, with concurrent transcriptional changes to a fetal pattern of gene expression), whereas activation of Akt is associated with the increased protein accretion in hypertrophy. Both ERK1/2 and Akt may promote myocyte survival. In the intact heart in vivo and in primary cultures of cardiac myocytes, mechanical strain induces hypertrophy, a process known as mechanotransduction, which may involve Ras, ERK1/2, and Akt. In this study, general and cardiospecific aspects of the regulation of Ras and Akt will be described. The various mechanisms through which mechanical strain might initiate Ras- or Akt-dependent signaling will be discussed. The overall conclusion is that although an involvement of Ras and Akt in mechanotransduction is likely, more work (particularly focusing on mechanoreception) needs to be undertaken before it is unequivocally established.

Expression profiling of Epo receptor in non-hematopoietic tissues: implications for a direct effect of rHuEpo on cardiac cells

Growing experimental evidence suggests that in addition to erythropoietin's (Epo) role in stimulating erythropoiesis, Epo may function in multiple tissues by preventing cellular apoptosis under stressful conditions such as hypoxia or exposure to cytotoxins and by stimulation of proliferation. EPO and EPOR knockout experiments in mice have demonstrated that the Epo/EpoR axis is essential for normal cardiac morphogenesis. While data suggests that there is an indirect effect of Epo on cardiac myocytes through increased hematocrit or other indirect effects, it is also possible that Epo acts directly on these cells. Therefore we have investigated the expression and function of EpoR in human cardiac cells. EpoR expression profiling in multiple murine, rat, monkey and human tissues using quantitative RT-PCR and RNAse protections (RPA's) identified EpoR transcripts in hematopoietic tissues, brain, kidney, adrenal gland, pancreas, liver and heart. More specifically in heart, EpoR transcripts were detected in monkey ventricle, atrium and septum. EpoR transcripts were also detected in primary human cardiac cell cultures, demonstrating that transcript detection was not due to contaminating hematopoietic cells. To determine whether transcription of EpoR correlated with expression of protein, immunolocalization experiments using a monoclonal antibody to human EpoR detected EpoR expression in human heart, located within cardiomyocytes, and within primary cardiac myocyte cultures. Staining was specific as demonstrated by reduced staining when the antibody was pre-incubated with soluble human EpoR. These data suggest that EpoR is expressed by cardiac cells and may suggest potential new functions of Epo in the heart. This may offer new options for the treatment of heart disease with erythropietic stimulating proteins such as darbepoetin alfa. Growing experimental evidence suggests that in addition to erythropoietin's (Epo) role in stimulating erythropoiesis, Epo may function in multiple tissues by preventing cellular apoptosis under stressful conditions such as hypoxia or exposure to cytotoxins and by stimulation of proliferation. EPO and EPOR knockout experiments in mice have demonstrated that the Epo/EpoR axis is essential for normal cardiac morphogenesis. While data suggests that there is an indirect effect of Epo on cardiac myocytes through increased hematocrit or other indirect effects, it is also possible that Epo acts directly on these cells. Therefore we have investigated the expression and function of EpoR in human cardiac cells. EpoR expression profiling in multiple murine, rat, monkey and human tissues using quantitative RT-PCR and RNAse protections (RPA's) identified EpoR transcripts in hematopoietic tissues, brain, kidney, adrenal gland, pancreas, liver and heart. More specifically in heart, EpoR transcripts were detected in monkey ventricle, atrium and septum. EpoR transcripts were also detected in primary human cardiac cell cultures, demonstrating that transcript detection was not due to contaminating hematopoietic cells. To determine whether transcription of EpoR correlated with expression of protein, immunolocalization experiments using a monoclonal antibody to human EpoR detected EpoR expression in human heart, located within cardiomyocytes, and within primary cardiac myocyte cultures. Staining was specific as demonstrated by reduced staining when the antibody was pre-incubated with soluble human EpoR. These data suggest that EpoR is expressed by cardiac cells and may suggest potential new functions of Epo in the heart. This may offer new options for the treatment of heart disease with erythropietic stimulating proteins such as darbepoetin alfa.

PKR's protective role in viral myocarditis

Reovirus-induced murine myocarditis provides an excellent model for the human disease. Previously, we showed that reovirus induction of and sensitivity to interferon-β (IFN-β) are important determinants of protection against cardiac damage. IFN-β induces a number of genes with antiviral activities, including the dsRNA-activated protein kinase, PKR. Once bound to viral dsRNA, PKR becomes activated and phosphorylates eukaryotic initiation factor-2α (eIF2α) leading to the cessation of host cell translation. Additionally, activated PKR can exert its antiviral effects by inducing phosphorylation of IκB, leading to the activation of the transcription factor NFκB and subsequent induction of IFN-β. Thus, activated PKR can both induce and be induced by IFN-β. Recently, numerous reports have shown PKR to be dispensable for both induction of IFN as well as protection against disease. However, both PKR's role in the heart in response to viral infection and its ability to prevent cardiac damage have gone largely unexplored. Here, we demonstrate PKR to be critical for viral induction of IFN-β in primary cardiac myocyte cultures. Additionally, we show that loss of PKR leads to an increase in virulence for both myocarditic and nonmyocarditic reoviruses. Finally, we demonstrate PKR to be critical for protection against reovirus-induced viral myocarditis.

Taurine transporter in primary cultured neonatal rat heart cells: a comparison between cardiac myocytes and nonmyocytes

In the present study, we examined the characteristics of the taurine transporter and the intracellular taurine content in cultured neonatal heart cells. Primary cultures of cardiac myocytes and cardiac fibroblasts (nonmyocytes) were prepared from 1-day-old Wistar rats. The parameters examined were: (a) intracellular taurine content by the HPLC method, (b) the expression levels of taurine transporter mRNA and protein using northern and western blot analysis, and (c) transporter activity determined by the uptake of 3 H -labeled taurine. The taurine content of myocytes was significantly higher (3-fold) than that of nonmyocytes. Taurine transporter mRNA was strongly expressed in both myocytes and nonmyocytes, whereas the magnitude [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression] of the transporter mRNA expressed in myocytes was lower than that in nonmyocytes. The expression level of transporter protein in myocytes was also lower than that of nonmyocytes. Uptake of radiolabeled taurine into monolayer cultures of heart cells was stimulated markedly by the presence of Na + in the medium, whereas this uptake was almost abolished in the absence of Na +. The Na +/taurine stoichiometry was 2:1 for both myocytes and nonmyocytes. Kinetic analysis showed that a single saturable system was involved in taurine uptake into both cell types. In myocytes, the apparent K m and V max values for the transporter were 20.7±0.5 μM and 1.07±0.01 nmol/10 6 cells/30 min, respectively. Similarly, those of nonmyocytes were 20.3±0.7 μM and 0.42±0.01 nmol/10 6 cells/30 min. These findings indicated that both myocytes and nonmyocytes expressed an identical taurine transporter with a Michaelis–Menten constant of 20–21 μM and that a higher taurine content in myocytes may be associated with a higher V max.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Interferon Regulatory Factor-1, Interferon-β, and Reovirus-Induced Myocarditis

Viral myocarditis is an important human disease, and reovirus-induced myocarditis in mice provides an excellent model to study direct viral damage to the heart. Previously, we showed that reovirus induction of and sensitivity to interferon-β (IFN-β) is an important determinant of viral pathogenicity in the heart and that the transcription factor interferon regulatory factor-3 (IRF-3) is required for reovirus induction of IFN-β in primary cardiac myocyte cultures. Given several lines of evidence suggesting a possible distinctive environment for IRFs in the heart, we have now focused on IRF-1. Previous studies demonstrated that viruses, double-stranded-RNA (dsRNA), and IFN-α/β can each induce IRF-1 and that IRF-1 plays a role in dsRNA, but perhaps not viral, induction of IFN-α/β. Importantly, none of these studies used a virus with a dsRNA genome (such as reovirus), none of them used a highly differentiated nonlymphoid cell type, and none of them addressed whether viral induction of IRF-1 is direct or is mediated through viral induction of IFN-β. Indeed, as recently as this year it has been assumed that viral induction of IRF-1 is direct. Here, we found that reovirus induced IRF-1 in primary cardiac myocyte cultures, but that IRF-1 was not required for reovirus induction of IFN-β. Surprisingly, we found that reovirus failed to induce IRF-1 in the absence of the IFN-α/β response. This provides the first evidence that viruses may not induce IRF-1 directly. Finally, nonmyocarditic reovirus strains induced more cardiac lesions in mice deficient for IRF-1 than they did in wildtype mice, directly demonstrating a protective role for IRF-1. Together, the results indicate that while IRF-1 is downstream of the IFN-β response, it plays an important protective role against viral myocarditis.

Optimal Range for Parvalbumin as Relaxing Agent in Adult Cardiac Myocytes: Gene Transfer and Mathematical Modeling

Parvalbumin (PV) has recently been shown to increase the relaxation rate when expressed in intact isolated cardiac myocytes via adenovirus gene transfer. We report here a combined experimental and mathematical modeling approach to determine the dose-response and the sarcomere length (SL) shortening-frequency relationship of PV in adult rat cardiac myocytes in primary culture. The dose-response was obtained experimentally by observing the PV-transduced myocytes at different time points after gene transfer. Calcium transients and unloaded mechanical contractions were measured. The results were as follows. At low estimated [PV] (∼0.01 mM), contractile parameters were unchanged; at intermediate [PV], relaxation rate of the mechanical contraction and the decay rate of the calcium transient increased with little effects on amplitude; and at high [PV] (∼0.1 mM), relaxation rate was further increased, but the amplitudes of the mechanical contraction and the calcium transient were diminished when compared with control myocytes. The SL shortening-frequency relationship exhibited a biphasic response to increasing stimulus frequency in controls (decrease in amplitude and re-lengthening time from 0.2 to 1.0 Hz followed by an increase in these parameters from 2.0 to 4.0 Hz). The effect of PV was to flatten this frequency response. This flattening effect was partly explained by a reduction in the variation in fractional binding of PV to calcium during beats at high frequency. In conclusion, experimental results and mathematical modeling indicate that there is an optimal PV range for which relaxation rate is increased with little effect on contractile amplitude and that PV effectiveness decreases as the stimulus frequency increases.

A potential cardioprotective role of hepatocyte growth factor in myocardial infarction in rats.

Cardiotrophic growth factors with anti-cell death actions on cardiac myocytes have gained attention for treatment of patients with myocardial infarction. Hepatocyte growth factor (HGF) plays a role in tissue repair and protection from injuries, however, the physiological role of HGF in the myocardium has not been well defined. We asked if HGF would afford to the infarcted myocardium. Mature cardiac myocytes prepared from adult rats expressed barely detectable levels of the c-Met/HGF receptor, however, c-Met receptor expression increased during cultivation, which meant that cardiac myocytes are potential targets of HGF. Addition of hydrogen peroxide remarkably decreased the number of viable mature cardiac myocytes in primary culture, whereas treatment with HGF enhanced survival of the cells subjected to the oxidant stress. Although very low levels of c-Met/HGF receptor and HGF mRNA expression were seen in normal rat hearts, both c-Met/HGF receptor and HGF mRNA levels rapidly increased to much higher levels than normal, when the rats were subjected to myocardial infarction. Immunohistochemical analysis of the c-Met receptor indicated that this receptor was expressed in cardiomyocytes localized in the border regions of the viable myocardium and in non-infarcted regions following myocardial infarction. The c-Met/HGF receptor is induced in cardiomyocytes following myocardial infarction and HGF exhibits protective effect on cardiomyocytes against oxidative stress. Our working hypothesis is that HGF may afford myocardial protection from myocardial infarction.