Introduction: Therapeutic agents like doxorubicin, an anthracycline antibiotic drug, are widely used in cancer chemotherapy. The use of doxorubicin is limited however by an increased risk of cardiac damage as a side effect, and an increased cancer cell drug resistance mediated by efflux drug transporters. Strategies are needed to protect the heart and still allow the benefits of drug treatment. “Basic” fibroblast growth factor-2 (FGF-2) is a multi-functional protein. It is angiogenic and cardioprotective against ischemia-reperfusion injury. FGF-2 can also regulate cancer cell drug resistance or sensitivity, however, so far, there is no evidence that FGF-2 protects against doxorubicin-induced cardiac damage through effects on efflux drug transporter levels or function. Aims: To investigate whether: (1) FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage; and if so whether (2) an effect on efflux drug transporters might contribute to this cardioprotection by FGF-2. Methods: Neonatal rat cardiac myocyte cultures were treated with doxorubicin in the absence or presence of pre-treatment with FGF-2. To assess cell damage: (i) culture medium was tested for lactate dehydrogenase (LDH) activity as an indication of plasma membrane disruption; (ii) cells were stained with fluorescent apoptosis and necrosis biomarkers as well as (iii) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and acridine orange to assess DNA fragmentation or compaction. The role of FGF receptor (FGFR) or protein kinase C (PKC) was addressed through use of inhibitors including SU5402, or chelerythrine as well as bisindomaleimide. Multidrug resistance gene 1a and 1b (MDR1a, 1b), multidrug resistance gene 2 (MDR2) and multidrug resistance-related protein 1 (MRP1) gene expression, as well as the function of MDRs and MRPs protein products were assessed by real-time reverse transcriptase-polymerase chain reaction (qPCR), as well as retention/extrusion of (fluorescent) doxorubicin/calcein in cardiac myocytes, respectively. Efflux drug transporter inhibitors, including 20 µM cyclosporine A (CsA), 2 µM verapamil and 1 µM Tariquidar (XR9576) were used to asssess for a direct effect of FGF-2 on transporter function. Fluorescence-activated cell sorting (FACS) was used to measure fluorescent doxorubicin/calcein levels inside treated cardiac myocytes. Results: Doxorubicin increased the incidence of programmed cell death, DNA damage, and lysosome and LDH activity, while decreasing cell number at 24 hours. FGF-2 prevented the detrimental effects of doxorubicin. In turn, the protective effects of FGF-2 were blocked in the presence of FGFR or PKC inhibitors. FGF-2 treatment significantly increased MDR1a, MDR1b, MDR2, MRP1 RNA levels by qPCR, and protein levels as assessed by function, and specifically extrusion of doxorubicin/calcein, in the presence of doxorubicin when compared to doxorubicin treatment alone. Furthermore, inhibition of efflux drug transporters with CsA and Tariquidar (XR9576) significantly reduced the ability of FGF-2 to protect against doxorubicin-induced damage; the beneficial effect of FGF-2 was completely blocked by pretreatment with verapamil. Conclusion(s): These data indicate for the first time that exogenous FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage, and implicate PKC and regulation of efflux transporter protein levels and/or function in the mechanism.

The main treatment for myocardial infarction is early reperfusion of ischemic tissue. Ischemia and reperfusion (IR) produces reactive oxygen species that oxidize membrane phospholipids. The production of oxidized lipids and their role on cell death in cardiac IR injury is unknown. Using in vitro model of IR, our goal was to identify oxidized phosphatidylcholines (OxPC) from cardiomyocytes, to determine their bioactivity on cardiomyocyte viability and mitochondrial permeability, and using an OxPC specific EO6 antibody inhibit OxPC activity on cardiomyocytes. Rat cardiomyocytes were exposed to IR and lipid extracts underwent lipidomic analysis with HPLC-MS/MS to quantitate 82 novel OxPC species. Cell viability and mitochondrial permeability were determined in vehicle control, non-oxidized control PC, and fragmented OxPC molecules. EO6 antibody was applied and cell viability was assessed. Cardiomyocytes under IR demonstrated increased relevant OxPCs particularly fragmented species. OxPC treatment resulted in loss of cardiomyocyte viability, increased mitochondrial permeability when compared to control. EO6 antibody blocked the loss of cardiomyocyte viability. We have shown for the first time that OxPCs are generated cardiomyocytes during IR and they have detrimental effects on cardiomyocyte viability. Additionally the EO6 antibody inhibits the bioactivity of the OxPCs on cardiomyocytes and could be part of a future treatment regimen.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

Drug-induced cardiotoxicity has resulted in a thorough evaluation of patient doses, treatments, and rehabilitation. One of the most commonly prescribed chemotherapeutic agents is cyclophosphamide. The active metabolite, acrolein, is one of the most potent inducers of cardiomyopathy. In this study, research was conducted on the H9c2 (2-1) cardiomyocyte cell line derived from the embryonic myocardium of rattus norvegicus to assess its competency for evaluation of the change in poly (ADP-ribose) polymerase (PARP) activity. The application of this model to study the effects of acrolein on PARP activation was chosen as an ideal determinant of cell damage produced by nitrogen mustards. To verify the legitimacy of this model, cardiomyocytes were exposed to acrolein in varying concentrations and time durations with a subsequent protein concentration measurement determined through the BCA Protein Assay. After the normalization of samples through volume adjustments and verification of sufficient protein, other aliquots were subjected to a PARP Assay in order to measure PARP activity. PARP was activated at exposure concentrations of 75 μM in all trials, with an average detection of 0.00569 ± 0.001 mU/200ng protein. Other concentrations showed varying degrees of PARP activation, verifying the model’s competency. PARP activation implies the potential use of this model for further research into targeted molecular therapy of PARP inhibition. Therefore, this model has the ability to be used as an assessment tool for the combined use of PARP inhibitors and chemotherapeutic agents; it can be useful for future research investigating the use and efficacy of PARP inhibitors in reducing drug-induced cardiotoxicity.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23471号 / 医博第4778号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The master´s thesis deals with research of viability and contraction measurement of cardiomyocytes. The work is divided into two main areas – theoretical and practical part. Theoretical part is aimed at electrophysiology of cardiomyocytes. Practical part includes detection of contractibility based on eccentricity in program Matlab. For research of viability, were used several media for example DMEM and MPRI.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal α-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120-180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.