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Recent discoveries of the molecular identity of mitochondrial Ca(2+) influx/efflux mechanisms have placed mitochondrial Ca(2+) transport at center stage in views of cellular regulation in various cell-types/tissues. Indeed, mitochondria in cardiac muscles also possess the molecular components for efficient uptake and extraction of Ca(2+). Over the last several years, multiple groups have taken advantage of newly available molecular information about these proteins and applied genetic tools to delineate the precise mechanisms for mitochondrial Ca(2+) handling in cardiomyocytes and its contribution to excitation-contraction/metabolism coupling in the heart. Though mitochondrial Ca(2+) has been proposed as one of the most crucial secondary messengers in controlling a cardiomyocyte’s life and death, the detailed mechanisms of how mitochondrial Ca(2+) regulates physiological mitochondrial and cellular functions in cardiac muscles, and how disorders of this mechanism lead to cardiac diseases remain unclear. In this review, we summarize the current controversies and discrepancies regarding cardiac mitochondrial Ca(2+) signaling that remain in the field to provide a platform for future discussions and experiments to help close this gap.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is the one of the GqPCR highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (

Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiomyocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed both in human heart failure (HF) and in animal HF models. However, it is still unclear which cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Recent reports suggest that Gq-protein coupled receptor (GqPCR) signaling pathways are critical for the development and progression of HF. Therefore, we hypothesize that GqPCR stimulation induces mitochondrial fragmentation and dysfunction, which initiates cardiomyocyte death. We found that protein kinase D (PKD) activated by GqPCR signaling was translocated to outer mitochondrial membrane (OMM) observed by Western blot analysis of cytosolic and mitochondria-enriched fractionated proteins and by live cell imaging of fluorescence resonance energy transfer (FRET). We also found that GqPCR-mediated PKD activation induced mitochondrial fragmentation, leading to increased reactive oxygen species (ROS) generation as well as increased mitochondrial permeability transition pore (mPTP) opening, which initiates apoptotic signaling activation and cardiomyocyte death. These morphological and functional changes in cardiac mitochondria were mediated via PKD-dependent phosphorylation of mitochondrial fission protein, Dynamin-Like Protein 1 (DLP1) at S637. Moreover, pretreatment with a novel potent PKD inhibitor CRT0066101 effectively inhibited GqPCR-mediated PKD translocation to OMM, DLP1 phosphorylation at S637, mitochondrial fragmentation, ROS generation and mPTP activation. In conclusion, we demonstrate that GqPCR stimulation induces mitochondrial fragmentation and dysfunction through PKD-dependent phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte injury. Thus, small-molecule PKD inhibitor may become a novel and potent therapeutic for preventing cardiac cell injury and death during HF.

We investigated regulation of various signal transduction pathways during oxidative stresses in the kidney of young and aged rats. Menadione-induced regulation of molecules in PI 3-kinase, MAPK, and AMPK pathways was determined in the young (2 months) and old (24 months) groups. PI 3-kinase activity and Akt phosphorylation were significantly reduced in the old compared with the young. PTEN tumor suppressor was also lower in its expression and phosphorylation levels in the old. Response of the molecules in PI 3-kinase pathway to menadione was minimized. In contrast, over 5-fold induction of ERK1/2 phosphorylation by menadione was observed in both groups. On the other hand, basal activities as well as menadione-induced activities of JNK1 and AMPK were higher in the old than in the young. While p27Kip1, p53, and p21Waf1 were slightly increased by menadione in both groups, the basal induction level in the old was considerably higher. In conclusion, the results suggest that the age-related down-regulation of PI 3-kinase/Akt pathway and up-regulation of JNK1, AMPK, and p53 pathways may be responsible for the increased susceptibility to oxidative stress.

Cardiac fibroblasts (CFs) are one o the most abundant cell types in the heart and play key roles in regulating myocardial physiological function and pathophysiological remodeling especially for the cardiac fibrosis. The levels of Angiotensin II (AT-II) are increased in the remodeling heart and Angiotensin signaling participates in pathological CF proliferation. It has been shown that CF proliferation may occur via the increased levels of cellular reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiac myofibroblast cell line H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhances mitochondrial Ca2+-induced mtSO generation in primary CFs, which can activate ROS-dependent proliferation signaling in primary CFs. First, we confirmed that AT-II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake assess by mitochondria-targeted Ca2+ biosensor in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT-II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. We also confirmed that AT-II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria-targeted antioxidant, Mito-tempo significantly inhibited AT-II-mediated activation of the mtSO production as well as proliferative pathway without changing the AT-II-induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation via MCU serves as an important regulator for the Ang II-induced proliferation in CFs.

Adrenoceptor stimulation is a key determinant of cardiac excitation–contraction coupling mainly through the activation of serine/threonine kinases. However, little is known about the role of protein tyrosine kinases (PTKs) activated by adrenergic signaling on cardiac excitation–contraction coupling. A cytoplasmic tyrosine residue in β(1)-adrenoceptor is estimated to regulate G(s)-protein binding affinity from crystal structure studies, but the signaling pathway leading to the phosphorylation of these residues is unknown. Here we show α(1)-adrenergic signaling inhibits b-adrenergically activated Ca(2+) current, Ca(2+) transients and contractile force through phosphorylation of tyrosine residues in β(1)-adrenoceptor by PTK. Our results indicate that inhibition of b-adrenoceptor-mediated Ca(2+) elevation by α(1)-adrenocep tor-PTK signaling serves as an important regulatory feedback mechanism when the catecholamine level increases to protect cardiomyocytes from cytosolic Ca(2+) overload.

Plasminogen activator inhibitor (PAI)-1 promotes development of asthma. PAI-1 mRNA and protein are markedly induced in activated mast cells (MCs), a major effector cell type in asthma. However, regulatory mechanisms of PAI-1 transcription in MCs are unknown. We present first evidence that PAI-1 is transcriptionally regulated in human MCs (hMCs). In addition to three enhancer regions, we demonstrated that the E-box at −566bp to −561bp is the negative regulatory element, and the specific and constitutive binding of the upstream stimulating factor-1 to this E-box is the key mechanism of the negative regulation of PAI-1 expression in hMCs.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and its activation during T cell receptor stimulation has recently been reported. In this study, we examined the role of AMPK in interleukin (IL)-2 production in T cells. Inhibition of AMPK by compound C, a specific inhibitor of AMPK or small interfering RNA of AMPKalpha1 suppressed IL-2 production in Jurkat T cells and peripheral blood lymphocytes stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. We then showed that AMPK inhibition reduced PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. Moreover, inhibition of AMPK suppressed transcriptional activation of NF-AT and AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that compound C inhibited PMA/Io-induced phosphorylation of p38, JNK, and GSK-3beta but not of ERK. These results suggest that AMPK mediates IL-2 production by regulating NF-AT and AP-1activation during T cell stimulation.

We examined the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), the dephosphorylated form of AICA ribotide (also termed “ZMP”), an intermediate of purine biosynthesis, on interleukin (IL)-2 production in T cells. AICAR inhibited IL-2 production in Jurkat T cells and peripheral blood lymphocytes activated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. Pretreatment with 5′-iodotubercidin, an adenosine kinase inhibitor, enhanced AICAR suppression of IL-2 production, suggesting that AICAR, not ZMP, is responsible for IL-2 suppression. We then showed that AICAR inhibited PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. AICAR inhibited DNA binding and transcriptional activation of NF-AT and to a lesser extent AP-1, but not NF-κB, in PMA/Io-activated Jurkat cells. Finally, we found that AICAR inhibited PMA/Io-induced phosphorylation of GSK-3 but not phosphorylation of ERK1/2, p38, and JNK. These results suggest that AICAR exerts its immunosuppressive effect in activated Jurkat cells by inhibiting GSK-3 phosphorylation and NF-AT activation.

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.

Mitochondria in cells undergo constant morphological changes mainly through fission and fusion. However, functional significance of mitochondrial fission and fusion is not fully understood. To test the importance of mitochondrial morphology in maintaining mitochondrial function, first, we used glucose-stimulated insulin secretion in pancreatic β-cells as an experimental model because insulin secretion upon elevated plasma glucose concentration requires intact mitochondrial function. Increased ATP production in mitochondria from glucose metabolism induces plasma membrane depolarization and subsequent increase of cytosolic Ca2+ triggers insulin exocytosis. We found that glucose stimulation of the β-cell line INS-1E induces transient mitochondrial shortening and recovery. Inhibiting mitochondrial fission by expressing the dominant-negative fission mutant DLP1-K38A abolished the dynamic change of mitochondrial morphology in glucose stimulation. Importantly, we discovered that abolition of the glucose-induced mitochondrial morphology change suppresses glucose-stimulated insulin secretion. Measuring respiration under fission inhibition showed an increase of mitochondrial uncoupling, and thus significantly diminished the mitochondrial ATP production in response to glucose stimulation. Further evaluation of mitochondrial membrane potential in primary hepatocytes revealed that inhibition of mitochondrial fission induces large-scale fluctuations of the potentiometric fluorescence in mitochondria within cells. Frequencies and intervals of the fluorescence oscillation were random and insensitive to inhibitors of anion channels and mitochondrial permeability, and superoxide scavenger. This suggests that the fission inhibition-induced fluctuation of the inner membrane potential is a previously unrecognized unique phenomenon. These observations demonstrate that inhibition of mitochondrial fission induces a large-scale fluctuation of the mitochondrial inner membrane potential, which is functionally reflected in mitochondrial uncoupling. Taken together, our findings indicate that mitochondrial fission plays a role in regulating the coupling efficiency of oxidative phosphorylation.

Background: Recent break-through discovery in the molecular identity of mitochondrial Ca2+ uniporter (MCU) opens the new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported in human sample mass spectroscopy, but the post translational modifications of MCU are completely unknown.Hypothesis: Tyrosine phosphorylation of MCU can modulate the mitochondrial Ca2+-uptake rate in cardiac cells.Methods: MCU was transiently or stably overexpressed in cardiac H9C2 cells. Tyrosine phosphorylation of MCU was detected by a general anti-phospho-tyrosine antibody. Mitochondrial Ca2+ concentration ([Ca2+]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam).Results: α1-adrenergic stimulation by phenylephrine enhanced the translocation of a Ca2+-dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) from cytosol to mitochondria followed by the increase in mitochondrial Pyk2 activity. Overexpressed MCU was exclusively localized at mitochondria and tyrosine residues in MCU were phosphorylated after Pyk2 activation. In addition, Pyk2 was bound to MCU at the basal condition and this interaction was enhanced by phenylephrine treatment. Moreover, Pyk2-dependent phosphorylation of MCU enhances MCU olgomerization observed by a conventional native PAGE. These effects were abolished by the co-transfection of kinase-dead Pyk2. In MCU-overexpressed cells, [Ca2+]m increased rapidly and reached to higher levels in response to cytosolic Ca2+ transients evoked by thapsigargin compared to non-transfected cells. Moreover, peak [Ca2+]m in MCU-overexpressed cells reached to much higher levels by phenylephrine pretreatment compared to non-treated cells.Conclusion: α1-adrenergic stimulation accelerates mitochondrial Ca2+ uptake through Pyk2-dependent direct phospholylation of MCU, which promotes the formation of tetrametric MCU channel pore. Our findings open up an exciting opportunity for investigating the first candidate cell signaling pathway for the MCU post translational modifications in cardiac cells.

Background:Cytosolic Ca2+ elevation is an important trigger for both mitochondrial dynamics and ATP generation in various cell types including cardiac myocytes. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Recent studies have identified additional Ca2+ uptake pathways, which exhibit different biophysical properties from MCU including skeletal-muscle type ryanodine receptor isoform1 (RyR1). However it is still unclear that which mitochondrial Ca2+ influx mechanism mainly participates in the regulation of mitochondrial morphology/dynamics and energetic functions in cardiac myocytes. Aim:To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology and function in cardiac cells. Methods:GFP- or non-tagged RyR1 was transiently or stably overexpressed in cardiac H9C2 cells. MCU and RyR2 are also expressed for comparisons. Mitochondrial localization of over-expressed RyR1 was observed by co-expression of mitochondrial matrix-targeted RFP (mtRFP) using confocal microscope and also quantified by Western blotting using mitochondria fractionation. Mitochondrial morphology was evaluated by the calculation of aspect ratio and form factor from mtRFP pictures. Mitochondrial Ca2+ concentration ([Ca2+]m) and ATP ([ATP]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam) and FRET-based indicators for ATP (ATeam), respectively.Results:Overexpressed RyR1 was partially localized in mitochondria, but the another RyR isoform RyR2 did not show any co-localization with mitochondria. Over-expression of RyR1 but not MCU and RyR2 showed the mitochondrial fragmentation. Fragmented mitochondria showed higher [Ca2+]m at peak and sustained Ca2+ transients compared to elongated mitochondria. RyR1 overexpressed cells had higher [ATP]m at the basal condition and showed more ATP production in response to [Ca2+]c elevation compared to non-transfected cells.Conclusion:Mitochondrial RyR1 strongly modulates mitochondrial morphology, Ca2+ influx and Ca2+-induced ATP production in cardiac cells.

Mitochondrial Ca2+ has a critical role for balancing cell survival and death. Ca2+ influx into mitochondrial matrix is mediated primarily by the mitochondrial calcium uniporter (MCU). However, the signaling pathways that regulate MCU channel functions via post-translational modifications of MCU are completely unknown. Here we show that adrenergic signaling induces MCU tyrosine phosphorylation and accelerates mitochondrial Ca2+ uptake in cardiac cells. Adrenergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine residues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane domains and 6 in the C-terminus), which are conserved across all eukaryotic species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphorylation candidate sites for protein tyrosine kinases using phosphorylation prediction programs. We mutated these tyrosine residues to phenylalanine and generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We confirmed that only two tyrosine sites were phosphorylated in response to adrenergic stimulation in situ using cell lines stably expressing MCU-YFs. In addition, overexpression of these MCU-YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation by thapsigargin, whereas wild-type MCU transfection dramatically accelerates mitochondrial Ca2+ uptake compared to non-transfected cells. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU can modulate its channel functions and regulate mitochondrial Ca2+ uptake.