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Mesenchymal Stem Cells Delivered With Alginate Hydrogel Improves Conduction at the Border Zone of Healed MI
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Heart Rhythm, Vol 7, No 11, November 2010
formation of functional stem cell-derived 3D myocardial tissue requires the support of non-myocytes.
Figure 3
MESENCHYMAL STEM CELLS DELIVERED WITH ALGINATE HYDROGEL IMPROVES CONDUCTION AT THE BORDER ZONE OF HEALED MI Nikhil C. Panda,* Sean Zuckerman,† KeKe Fan,* Devi Gopinath,* David S. Rosenbaum,* Marc S. Penn,‡ J. Kevin Donahue,* Eben Alsberg,† Kenneth R. Laurita*. *MetroHealth Campus of CWRU, Cleveland, OH, †Department of Biomedical Engineering, CWRU, Cleveland, OH, ‡Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, OH Background: Mesenchymal stem cells (MSCs) for the treatment of myocardial infarction (MI) have been associated with an electrophysiological benefit; however, limited retention and survival of MSCs can significantly reduce efficacy. We hypothesized that a naturally derived biopolymer alginate hydrogel acting as a tissue scaffold will enhance retention and survival of MSCs injected at the border zone of healed MI and improve conduction. Methods and Results: Endovascular balloon inflation of the mid-left anterior descending artery for 2.5 hours was performed on female Yorkshire pigs (n⫽6). After four weeks, 10 million DiI labeled allogeneic pig MSCs suspended in PBS (control), 1%, or 2% RGD-modified alginate were injected epicardially into the MI border zone. Injection and pacing sites were marked with sutures. Conduction time (CT) during steady state pacing was measured in the epicardial border zone just before cells were injected. After 9-21 days, CT was measured again from the same location. MSC engraftment was quantified by measuring DiI fluorescence in tissue samples taken from all injection sites. Confocal immunofluorescence (IF) demonstrated that MSCs survive and express Cx43 when suspended in either PBS, 1%, or 2% alginate. We found that 2% alginate significantly increased MSC retention and survival compared to 1% alginate (126%, P⬍.01) and PBS (756%, P⬍.001). CT in the infarct border zone was shorter after MSC therapy compared to before, at pacing cycle lengths of 300 ms (⫺9⫾4%, P⬍.05) and 350 ms (⫺4⫾1%, P⬍.05). Conclusions: Alginate hydrogel significantly improves the retention and survival of MSCs that are directly injected into the border zone of healed MI. Moreover, CT in the border zone was significantly improved after the injection of MSCs suspended in hydrogel. Therefore, MSCs suspended in alginate hydrogel may be an effective treatment strategy for improving electrophysiological dysfunction associated with healed MI.
NOVEL BLOCKERS OF T-TYPE CALCIUM CHANNELS MODIFY GATING Pamela Bergson,* Tiffany Hu,* Victor N. Uebele,† John J. Renger,† Dorothy A. Hanck*. *Department of Medicine, Section of Cardiology, University of Chicago, Chicago, IL, †Department of Depression and Circadian Disorders, Merck Research Laboratories, West Point, PA
Background: T-type calcium channels are a subfamily of three voltagegated ion channels (CaV3.1, CaV3.2 and CaV3.3). They are characterized by their small conductance and low threshold of activation. T-type calcium channels are expressed in the developing heart. In adults, T-type channels are present in the vasculature but appear to be absent from normal human cardiac myocytes. T-type channels may be re-expressed in failing hearts during remodeling. They represent an important drug target, both to enable basic research to determine function and as therapeutic targets. Although no specific blockers are currently available for clinical use, several compounds have recently been identified that block calcium entry into cells expressing T-type channels. We have examined the mechanisms by which three of these drugs, TTA-P2, TTA-A2 and TTA-Q4, block T-type channels. Methods and Results: T-type channel currents were recorded from HEK 293 cells expressing CaV3.1 using whole cell patch clamp. When the holding potential was ⫺110 mV, the TTAs blocked resting T-type channels with sub-micromolar affinity (ED50⫽40 nM for TTA-P2, 890 nM for TTA-A2 and 560 nM for TTA-Q4). Block by TTA-P2 was not voltage dependent. The extent, but not the kinetics, of block by the other two compounds was strongly voltage dependent. We recorded gating currents in solutions designed to minimize contamination by ionic current. TTA-A2 and TTA-Q4 inhibited the movement of OFF gating charge. Conclusions: These data indicate that, although all three drugs block T-type channels with unusual potency and specificity, their mechanisms of block differ. TTA-A2 and TTA-Q4 modify the gating of T-type calcium channels and may be useful for the study of T-type channel gating. Understanding the differences between these compounds will provide valuable information about T-type channels and may assist in the development of potential therapeutic drugs. Support: T32HL007381 (PB), RO1HL065680 (DAH).
DIABETIC RABBIT HEARTS DISPLAY IMPAIRED CARDIAC PROPAGATION PROPERTIES S.V. Pandit, A. Mitra, M. Deo, S. Mironov, W. Cawthorn,* C.L. Stables, J. Jalife. Department of Internal Medicine-Cardiology and ⴱDepartment of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI Background: The electrophysiological alterations occurring in diabetic hearts remain unclear. Our objective was to study cardiac repolarization and propagation in diabetic hearts under normal conditions and during variations in extracellular K⫹ ([K⫹]o: hypokalemia, hyperkalemia). Methods: We created an alloxan-injected, type-1 model of diabetes in rabbits. Optical mapping was used to record electrical activity in Langendorff-perfused hearts from control and diabetic rabbits in normokalemia ([K⫹]o⫽4 mM), hypokalemia ([K⫹]o⫽2 mM), and hyperkalemia ([K⫹]o⫽12 mM). Concomitant computer simulations were performed to help interpret experimental findings. Results: The hearts from diabetic rabbits were mapped 123⫾8.42 days after induction of diabetes (n⫽7). Diabetic rabbits displayed higher glucose levels compared to normal rabbits (428⫾22.61 mg/dL vs. 159.2⫾12.25 mg/dL; n⫽6, P⬍.05). At a pacing cycle length of 250 ms (4 Hz), the cardiac action potential duration at 70% (APD70) was not different between control and diabetic hearts in normokalemia, hypokalemia, or hyperkalemia. In contrast, the conduction velocity at 4 Hz pacing (CV4Hz) at [K⫹]o⫽4 mM was slightly but significantly slower by ⬇13% in diabetic hearts compared to controls [CV4Hz ⫽ 0.53⫾0.02 m/s in control (n⫽8) vs. 0.46⫾0.02 m/s in diabetes (n⫽7), P⬍.05]. Similarly, CV4Hz in hypokalemia was significant slower by ⬇17% in diabetic hearts compared to controls. The difference in conduction velocities was further enhanced in hyperkalemic conditions ([K⫹]o⫽12 mM). We were able to obtain 1:1 pacing at 4 Hz in 5/7 control hearts and in 3/7 diabetic hearts in hyperkalemia. The CV was slower by ⬇33% in diabetic hearts in hyperkalemia [CV4Hz ⫽ 0.33⫾0.02 m/s in control (n⫽5) vs. 0.22⫾0.02 m/s in diabetes (n⫽3), P⬍.05]. Arrhythmogenic patterns (conduction block, wavebreaks,
Heart Rhythm, Vol 7, No 11, November 2010
formation of functional stem cell-derived 3D myocardial tissue requires the support of non-myocytes.
Figure 3
MESENCHYMAL STEM CELLS DELIVERED WITH ALGINATE HYDROGEL IMPROVES CONDUCTION AT THE BORDER ZONE OF HEALED MI Nikhil C. Panda,* Sean Zuckerman,† KeKe Fan,* Devi Gopinath,* David S. Rosenbaum,* Marc S. Penn,‡ J. Kevin Donahue,* Eben Alsberg,† Kenneth R. Laurita*. *MetroHealth Campus of CWRU, Cleveland, OH, †Department of Biomedical Engineering, CWRU, Cleveland, OH, ‡Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, OH Background: Mesenchymal stem cells (MSCs) for the treatment of myocardial infarction (MI) have been associated with an electrophysiological benefit; however, limited retention and survival of MSCs can significantly reduce efficacy. We hypothesized that a naturally derived biopolymer alginate hydrogel acting as a tissue scaffold will enhance retention and survival of MSCs injected at the border zone of healed MI and improve conduction. Methods and Results: Endovascular balloon inflation of the mid-left anterior descending artery for 2.5 hours was performed on female Yorkshire pigs (n⫽6). After four weeks, 10 million DiI labeled allogeneic pig MSCs suspended in PBS (control), 1%, or 2% RGD-modified alginate were injected epicardially into the MI border zone. Injection and pacing sites were marked with sutures. Conduction time (CT) during steady state pacing was measured in the epicardial border zone just before cells were injected. After 9-21 days, CT was measured again from the same location. MSC engraftment was quantified by measuring DiI fluorescence in tissue samples taken from all injection sites. Confocal immunofluorescence (IF) demonstrated that MSCs survive and express Cx43 when suspended in either PBS, 1%, or 2% alginate. We found that 2% alginate significantly increased MSC retention and survival compared to 1% alginate (126%, P⬍.01) and PBS (756%, P⬍.001). CT in the infarct border zone was shorter after MSC therapy compared to before, at pacing cycle lengths of 300 ms (⫺9⫾4%, P⬍.05) and 350 ms (⫺4⫾1%, P⬍.05). Conclusions: Alginate hydrogel significantly improves the retention and survival of MSCs that are directly injected into the border zone of healed MI. Moreover, CT in the border zone was significantly improved after the injection of MSCs suspended in hydrogel. Therefore, MSCs suspended in alginate hydrogel may be an effective treatment strategy for improving electrophysiological dysfunction associated with healed MI.
NOVEL BLOCKERS OF T-TYPE CALCIUM CHANNELS MODIFY GATING Pamela Bergson,* Tiffany Hu,* Victor N. Uebele,† John J. Renger,† Dorothy A. Hanck*. *Department of Medicine, Section of Cardiology, University of Chicago, Chicago, IL, †Department of Depression and Circadian Disorders, Merck Research Laboratories, West Point, PA
Background: T-type calcium channels are a subfamily of three voltagegated ion channels (CaV3.1, CaV3.2 and CaV3.3). They are characterized by their small conductance and low threshold of activation. T-type calcium channels are expressed in the developing heart. In adults, T-type channels are present in the vasculature but appear to be absent from normal human cardiac myocytes. T-type channels may be re-expressed in failing hearts during remodeling. They represent an important drug target, both to enable basic research to determine function and as therapeutic targets. Although no specific blockers are currently available for clinical use, several compounds have recently been identified that block calcium entry into cells expressing T-type channels. We have examined the mechanisms by which three of these drugs, TTA-P2, TTA-A2 and TTA-Q4, block T-type channels. Methods and Results: T-type channel currents were recorded from HEK 293 cells expressing CaV3.1 using whole cell patch clamp. When the holding potential was ⫺110 mV, the TTAs blocked resting T-type channels with sub-micromolar affinity (ED50⫽40 nM for TTA-P2, 890 nM for TTA-A2 and 560 nM for TTA-Q4). Block by TTA-P2 was not voltage dependent. The extent, but not the kinetics, of block by the other two compounds was strongly voltage dependent. We recorded gating currents in solutions designed to minimize contamination by ionic current. TTA-A2 and TTA-Q4 inhibited the movement of OFF gating charge. Conclusions: These data indicate that, although all three drugs block T-type channels with unusual potency and specificity, their mechanisms of block differ. TTA-A2 and TTA-Q4 modify the gating of T-type calcium channels and may be useful for the study of T-type channel gating. Understanding the differences between these compounds will provide valuable information about T-type channels and may assist in the development of potential therapeutic drugs. Support: T32HL007381 (PB), RO1HL065680 (DAH).
DIABETIC RABBIT HEARTS DISPLAY IMPAIRED CARDIAC PROPAGATION PROPERTIES S.V. Pandit, A. Mitra, M. Deo, S. Mironov, W. Cawthorn,* C.L. Stables, J. Jalife. Department of Internal Medicine-Cardiology and ⴱDepartment of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI Background: The electrophysiological alterations occurring in diabetic hearts remain unclear. Our objective was to study cardiac repolarization and propagation in diabetic hearts under normal conditions and during variations in extracellular K⫹ ([K⫹]o: hypokalemia, hyperkalemia). Methods: We created an alloxan-injected, type-1 model of diabetes in rabbits. Optical mapping was used to record electrical activity in Langendorff-perfused hearts from control and diabetic rabbits in normokalemia ([K⫹]o⫽4 mM), hypokalemia ([K⫹]o⫽2 mM), and hyperkalemia ([K⫹]o⫽12 mM). Concomitant computer simulations were performed to help interpret experimental findings. Results: The hearts from diabetic rabbits were mapped 123⫾8.42 days after induction of diabetes (n⫽7). Diabetic rabbits displayed higher glucose levels compared to normal rabbits (428⫾22.61 mg/dL vs. 159.2⫾12.25 mg/dL; n⫽6, P⬍.05). At a pacing cycle length of 250 ms (4 Hz), the cardiac action potential duration at 70% (APD70) was not different between control and diabetic hearts in normokalemia, hypokalemia, or hyperkalemia. In contrast, the conduction velocity at 4 Hz pacing (CV4Hz) at [K⫹]o⫽4 mM was slightly but significantly slower by ⬇13% in diabetic hearts compared to controls [CV4Hz ⫽ 0.53⫾0.02 m/s in control (n⫽8) vs. 0.46⫾0.02 m/s in diabetes (n⫽7), P⬍.05]. Similarly, CV4Hz in hypokalemia was significant slower by ⬇17% in diabetic hearts compared to controls. The difference in conduction velocities was further enhanced in hyperkalemic conditions ([K⫹]o⫽12 mM). We were able to obtain 1:1 pacing at 4 Hz in 5/7 control hearts and in 3/7 diabetic hearts in hyperkalemia. The CV was slower by ⬇33% in diabetic hearts in hyperkalemia [CV4Hz ⫽ 0.33⫾0.02 m/s in control (n⫽5) vs. 0.22⫾0.02 m/s in diabetes (n⫽3), P⬍.05]. Arrhythmogenic patterns (conduction block, wavebreaks,