Mesenchymal stem cells stimulate protective genetic reprogramming of injured cardiac ventricular myocytes.

Since massive irreversible loss of cardiac myocytes occurs following myocardial injury, injection of human mesenchymal stem cells (hMSCs) has emerged as a promising therapeutic intervention. Despite the growing enthusiasm for this approach, the understanding of how hMSCs evoke cardiac improvement is ever more controversial. The present study critically tests hypothesis that hMSCs provide specific benefit directly to damaged ventricular myocytes. Cultures of neonatal mouse ventricular cardiac myocytes (nMCM) were subjected to two distinct acute stress protocols; incubations with either endotoxin, lipopolysaccharide (LPS) or toxic cytokine, IL-1ß. Myocyte injury was assessed in intracellular Ca(2+) signaling assays in fluo-3-loaded nMCMs that were imaged with high temporal resolution by fluorescent microscopy. Following LPS or IL-1ß treatment there was profound myocyte injury, manifest by chaotic [Ca(2+)](i) handling, quantified as a 3- to 5-fold increase in spontaneous [Ca(2+)](i) transients. Antibody neutralization experiments reveal such damage is mediated in part by interleukin-18 and not by tumor necrosis factor-α (TNF-α). Importantly, normal [Ca(2+)](i) signaling was preserved when cardiomyocytes were co-cultured with hMSCs. Since normal [Ca(2+)](i) handling was maintained in transwell cultures, where nMCMs and hMSCs were separated by a permeable membrane, a protective paracrine signaling cascade is operable. hMSCs provoke a genetic reprogramming of cardiomyocytes. LPS provokes release of TNFα from nMCMs which is blocked by hMSCs grown in co- or transwell cultures. Consistent with cytokine release, flow cytometry analyses reveal that hMSCs also block the LPS- and IL-1ß-dependent activation of cardiac transcription factor, NF-κB. Importantly, hMSC-conditioned medium restores normal Ca(2+) signaling in LPS- and IL-1ß-damaged nMCMs. These results reveal new evidence that hMSCs elicit protective and reparative effects on cardiac tissue through molecular reprogramming of the cardiac myocytes themselves. Thus these studies provide novel new insight into the cellular and molecular mechanisms that underlie the therapeutic benefit of hMSCs in the setting of heart failure. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Novel function of cardiac protein kinase D1 as a dynamic regulator of Ca2+ sensitivity of contraction.

Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.

Proteomic studies of PP2A-B56gamma1 phosphatase complexes reveal phosphorylation-regulated partners in cardiac local signaling.

Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56gamma1, expressed in heart cells. As the role of PP2A/B56gamma1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation. The results reveal multiple interactors that are localized in strategic cardiac sites to participate in Ca2+ homeostasis and gene expression, exemplified by the Ca pump, SERCA2a, and the splicing factor ASF/SF2. These results are corroborated by confocal imaging where adenovirally overexpressed B56gamma1 is found in z-line/t-tubule region and nuclear speckles. Importantly, overexpression of B56gamma1 in cultured myocytes dramatically impairs cell contractility. These results provide a global view of B56gamma1-regulated local signaling and heart function.

Molecular basis for PP2A regulatory subunit B56alpha targeting in cardiomyocytes.

Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of beta-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56alpha is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56alpha are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56alpha for ankyrin-B association as a 13 residue motif in the B56alpha COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B(+/-) cardiomyocytes results in disorganized distribution of B56alpha that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides.

Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma.

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

Inhibitor-kappaB kinase-beta regulates LPS-induced TNF-alpha production in cardiac myocytes through modulation of NF-kappaB p65 subunit phosphorylation.

TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-alpha production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappaB signaling in TNF-alpha gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.

A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes.

Protein phosphatase 2A (PP2A) is widely distributed in heart tissues, yet its precise cellular functions are poorly understood. This study is based on the notion that PP2A action is governed by interactions of the core enzyme with B targeting/regulatory subunits. The subcellular localizations of two B subunits, B56alpha and B56gamma1, were assessed using adenovirus-driven expression of epitope-tagged (hemagglutinin, HA) in cultured neonatal and adult rat ventricular myocytes. Confocal imaging revealed that HA-B56alpha was excluded from the nucleus and decorated striated structures, whereas HA-B56gamma1 was principally found in the nucleus. Precise immunolabeling studies showed that B56gamma1 was concentrated in intranuclear structures known as nuclear speckles, macromolecular structures that accumulate transcription and splicing factors. Western blot analyses revealed that overexpression of either B subunit had no effect on the levels of other PP2A subunits in cultured neonatal cardiac cells. However, overexpression of only B56gamma1 increased whole cell PP2A activity by 40% when measured in cell extracts. Finally, B56gamma1 did not alter global gene expression or expression of hypertrophic gene markers such as alpha-skeletal actin. However, morphometric analyses of confocal images revealed that B56gamma1 alters the dynamic assembly/disassembly process of nuclear speckles in heart cells. These studies provide new insight into mechanisms of PP2A targeting in the subnuclear architecture in cardiomyocytes and into the role of this phosphatase in nuclear signaling.

Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes.

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I(Ca), the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal'I(Ca) in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V(0.5) (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I(Ca) when added after calyculin A. However, by itself, staurosporine decreased I(Ca) throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 microm in the pipette) and H89 (1 microm) failed to reduce basal I(Ca) or to block the calyculin A-evoked increase in I(Ca). Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba(2+) was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 microm) and Gö 6976 (200 nm) decreased steady-state I(Ca) and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I(Ca). However, PP1 is important, as dialysis with 2 microm okadaic acid or 500 nm inhibitor-2 mimicked the increases in I(Ca) seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I(Ca).

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Endotoxin stress-response in cardiomyocytes: NF-kappaB activation and tumor necrosis factor-alpha expression.

Although tumor necrosis factor (TNF)-alpha is implicated in numerous cardiac pathologies, the intracellular events leading to its production by heart cells are largely unknown. The goal of the present study was to identify the role of the transcription factor nuclear factor (NF)-kappaB in this process. Among the many inducers of TNF-alpha expression in myeloid cells, only lipopolysaccharide (LPS) led to its induction in cultured neonatal myocytes. LPS also activated the NF-kappaB pathway, as evidenced by the degradation of the inhibitory protein IkappaB and the appearance of NF-kappaB-binding complexes in nuclear extracts. Furthermore, inhibitors of NF-kappaB activation, such as lactacystin, MG132, and pyrrolidine dithiocarbamate, were found to completely block the production of TNF-alpha in response to LPS stimulation, indicating a requirement of NF-kappaB for TNF-alpha expression. However, interleukin-1beta and phorbol 12-myristate 13-acetate also activated NF-kappaB but did not evoke TNF-alpha expression, revealing that this factor is not sufficient for cytokine production. Detailed examination of the NF-kappaB cascade revealed that cardiac cells displayed a unique pattern of IkappaB degradation in response to LPS, with IkappaBbeta but not IkappaBalpha being degraded upon stimulation. Additionally, two specific p65-containing DNA-binding complexes were observed in the nuclear extracts of neonatal cardiomyocytes: an inducible complex that is necessary for TNF-alpha expression and a constitutive species. Taken together, these results reveal that NF-kappaB is not only involved in cytokine production but also may be linked to other pathways that subserve a constitutive, protective mechanism for the heart cell.