eb5be901c744b0ed94c4192f79991233 264736.pdf 2e70b7c5e5932de156f95309538112274248d087 264736.pdf f27824aa651a965c33beaa2e412805a8560f211859bd027c8f92fa26b2a1be5b 264736.pdf Title: Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling Subject: Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the -agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to -adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation). Keywords: atrial fibrillation (AF); cAMP-dependent regulation; protein kinase A (PKA); L-type calcium current (ICa,L); transient inward current (ITI); patch-clamp Author: Franziska Reinhardt, Kira Beneke, Nefeli Grammatica Pavlidou, Lenard Conradi, Hermann Reichenspurner, Leif Hove-Madsen and Cristina E. Molina Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Nov 5 12:50:31 2021 CET ModDate: Fri Nov 5 12:54:20 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2207385 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 10 0 UNJBAV+EURM10 Type 1 Builtin yes yes no 15 0 OYYMZB+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 BQGXVZ+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 RBQDYP+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 VDTJSN+CMSY10 Type 1 Builtin yes yes no 32 0 PFKOHU+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 40 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 62 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 66 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 70 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 91 0 DNNNME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 101 0 DNNNMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 104 0 DNNNNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 107 0 DNNNNH+PalatinoLinotype TrueType WinAnsi yes yes no 113 0 DNNNMD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 123 0 DNNNME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 129 0 DNNNMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 132 0 DNNNNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 135 0 DNNNNH+PalatinoLinotype TrueType WinAnsi yes yes no 141 0 DNNOIF+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 144 0 DNNNME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 154 0 DNNNMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 157 0 DNNNNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 160 0 DNNNNH+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 DNNNMD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 176 0 DNNNME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 182 0 DNNNMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 185 0 DNNNNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 188 0 DNNNNH+PalatinoLinotype TrueType WinAnsi yes yes no 194 0 DNNOIF+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 197 0 DNNNMD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 207 0 DNNNME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 213 0 DNNNMG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 216 0 DNNNNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 219 0 DNNNNH+PalatinoLinotype TrueType WinAnsi yes yes no 225 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:21:53 CEST RepresentationInformation: 264736.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:59:22 CEST Size: 2207385 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 524 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Human Atrial Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Isolation and Culture of Human Atrial Myocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Patch-Clamp Technique Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Confocal Calcium Imaging Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Fluorescence Resonance Energy Transfer (FRET)-Based Live Cell Imaging of Intracellular cAMP Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Data Analysis and Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: cAMP-Dependent Regulation of L-Type Ca2+ Current and Spontaneous SR Ca2+ Release Show Opposite Changes in Atrial Fibrillation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: CaMKII Inhibition Reduces ICa,L and Ca2+ Sparks but Not ITI Frequency Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: -Adrenergic Stimulates ICa,L Equally in Ctl and AF Patients but Has a Relatively Smaller Effect on ITI Frequency in AF Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: -Adrenergic Stimulation Produces Different Increase in cAMP Levels in Ctl and AF in Different Cellular Compartments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Carvedilol, but Not 1-Blocker Treatment, Attenuates PKA Dependent Increase of ITI in AF Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: PKA-Dependent Modulation of Ca2+ Homeostasis in AF Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: CaMKII-Dependent Regulation of Ca2+ Sparks in AF Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Key Ca2+-Handling Compartments Are Regulated by Specific cAMP Microdomains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Study Limitations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling Author: Franziska Reinhardt, Kira Beneke, Nefeli Grammatica Pavlidou, Lenard Conradi, Hermann Reichenspurner, Leif Hove-Madsen and Cristina E. Molina Subject: Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the -agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to -adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation). Keywords: atrial fibrillation (AF); cAMP-dependent regulation; protein kinase A (PKA); L-type calcium current (ICa,L); transient inward current (ITI); patch-clamp Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Nov 05 12:50:31 CET 2021 ModDate: Fri Nov 05 12:54:20 CET 2021 ID: 0x5cccd87156b6e8849db0f2fcf7993d41, 0x5cccd87156b6e8849db0f2fcf7993d41 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VDTJSN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: VDTJSN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: OYYMZB+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: OYYMZB+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: BQGXVZ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: BQGXVZ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OPZDHV+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: OPZDHV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CRJHCU+PazoMath-Italic FontSubset: true FirstChar: 98 LastChar: 98 FontDescriptor: FontName: CRJHCU+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: RBQDYP+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: RBQDYP+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UNJBAV+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: UNJBAV+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 122, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 388, 647, 400, 661 Annotation: Subtype: Link Rect: 55, 358, 154, 367 Annotation: Subtype: Link Rect: 35, 348, 78, 355 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 391, 130, 398, 139 ActionDest: 239 Annotation: Subtype: Link Rect: 401, 130, 408, 139 ActionDest: 239 Annotation: Subtype: Link Rect: 366, 118, 373, 127 ActionDest: 239 Annotation: Subtype: Link Rect: 373, 118, 380, 127 ActionDest: 239 Annotation: Subtype: Link Rect: 381, 118, 388, 127 ActionDest: 239 Annotation: Subtype: Link Rect: 349, 105, 356, 114 ActionDest: 239 Annotation: Subtype: Link Rect: 548, 80, 555, 89 ActionDest: 242 Annotation: Subtype: Link Rect: 104, 17, 244, 28 Annotation: Subtype: Link Rect: 419, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 458, 87, 477 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 397, 724, 404, 733 ActionDest: 239 Annotation: Subtype: Link Rect: 405, 724, 412, 733 ActionDest: 242 Annotation: Subtype: Link Rect: 415, 724, 427, 733 ActionDest: 242 Annotation: Subtype: Link Rect: 219, 699, 226, 708 ActionDest: 239 Annotation: Subtype: Link Rect: 227, 699, 234, 708 ActionDest: 239 Annotation: Subtype: Link Rect: 234, 699, 241, 708 ActionDest: 239 Annotation: Subtype: Link Rect: 434, 636, 441, 645 ActionDest: 239 Annotation: Subtype: Link Rect: 398, 611, 410, 620 ActionDest: 242 Annotation: Subtype: Link Rect: 259, 586, 266, 595 ActionDest: 239 Annotation: Subtype: Link Rect: 267, 586, 279, 595 ActionDest: 242 Annotation: Subtype: Link Rect: 543, 561, 555, 570 ActionDest: 242 Annotation: Subtype: Link Rect: 473, 548, 485, 557 ActionDest: 242 Annotation: Subtype: Link Rect: 542, 511, 549, 520 ActionDest: 239 Annotation: Subtype: Link Rect: 550, 511, 557, 520 ActionDest: 239 Annotation: Subtype: Link Rect: 291, 498, 298, 507 ActionDest: 242 Annotation: Subtype: Link Rect: 514, 118, 521, 130 ActionDest: 82 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 382, 342, 394, 351 ActionDest: 242 Annotation: Subtype: Link Rect: 394, 342, 406, 351 ActionDest: 242 Annotation: Subtype: Link Rect: 549, 65, 556, 74 ActionDest: 239 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 469, 660, 476, 669 ActionDest: 242 Annotation: Subtype: Link Rect: 208, 634, 220, 643 ActionDest: 242 Annotation: Subtype: Link Rect: 200, 383, 212, 392 ActionDest: 242 Annotation: Subtype: Link Rect: 369, 332, 381, 342 ActionDest: 242 Annotation: Subtype: Link Rect: 439, 332, 451, 342 ActionDest: 242 Annotation: Subtype: Link Rect: 531, 332, 543, 342 ActionDest: 242 Annotation: Subtype: Link Rect: 359, 157, 371, 166 ActionDest: 242 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 354, 693, 361, 702 ActionDest: 239 Annotation: Subtype: Link Rect: 362, 693, 369, 702 ActionDest: 239 Annotation: Subtype: Link Rect: 369, 693, 376, 702 ActionDest: 239 Annotation: Subtype: Link Rect: 200, 602, 207, 614 ActionDest: 95 Annotation: Subtype: Link Rect: 429, 590, 436, 602 ActionDest: 95 Annotation: Subtype: Link Rect: 458, 577, 465, 589 ActionDest: 95 Annotation: Subtype: Link Rect: 420, 565, 427, 578 ActionDest: 95 Annotation: Subtype: Link Rect: 319, 399, 326, 411 ActionDest: 117 Annotation: Subtype: Link Rect: 299, 374, 306, 386 ActionDest: 117 Annotation: Subtype: Link Rect: 468, 361, 475, 375 ActionDest: 117 Annotation: Subtype: Link Rect: 239, 275, 246, 287 ActionDest: 148 Annotation: Subtype: Link Rect: 435, 250, 442, 262 ActionDest: 148 Annotation: Subtype: Link Rect: 544, 212, 551, 224 ActionDest: 148 Annotation: Subtype: Link Rect: 466, 200, 472, 212 ActionDest: 148 Annotation: Subtype: Link Rect: 304, 87, 311, 99 ActionDest: 148 Annotation: Subtype: Link Rect: 441, 74, 448, 88 ActionDest: 148 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 353, 707, 360, 719 ActionDest: 148 Page: Sequence: 7 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 450, 643, 457, 655 ActionDest: 170 Annotation: Subtype: Link Rect: 411, 581, 418, 593 ActionDest: 170 Annotation: Subtype: Link Rect: 446, 184, 458, 193 ActionDest: 242 Annotation: Subtype: Link Rect: 490, 106, 497, 118 ActionDest: 170 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 357, 654, 365, 666 ActionDest: 201 Annotation: Subtype: Link Rect: 258, 641, 265, 655 ActionDest: 201 Annotation: Subtype: Link Rect: 371, 641, 378, 655 ActionDest: 201 Annotation: Subtype: Link Rect: 497, 641, 504, 655 ActionDest: 201 Annotation: Subtype: Link Rect: 407, 591, 414, 603 ActionDest: 201 Annotation: Subtype: Link Rect: 527, 579, 534, 591 ActionDest: 201 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 317, 566, 324, 575 ActionDest: 239 Annotation: Subtype: Link Rect: 325, 566, 332, 575 ActionDest: 239 Annotation: Subtype: Link Rect: 332, 566, 344, 575 ActionDest: 242 Annotation: Subtype: Link Rect: 426, 541, 438, 550 ActionDest: 242 Annotation: Subtype: Link Rect: 439, 541, 451, 550 ActionDest: 242 Annotation: Subtype: Link Rect: 543, 541, 555, 550 ActionDest: 242 Annotation: Subtype: Link Rect: 383, 529, 395, 537 ActionDest: 242 Annotation: Subtype: Link Rect: 493, 500, 500, 513 ActionDest: 95 Annotation: Subtype: Link Rect: 219, 441, 226, 450 ActionDest: 239 Annotation: Subtype: Link Rect: 226, 441, 233, 450 ActionDest: 239 Annotation: Subtype: Link Rect: 234, 441, 241, 450 ActionDest: 239 Annotation: Subtype: Link Rect: 340, 441, 347, 450 ActionDest: 239 Annotation: Subtype: Link Rect: 348, 441, 360, 450 ActionDest: 242 Annotation: Subtype: Link Rect: 500, 428, 507, 437 ActionDest: 239 Annotation: Subtype: Link Rect: 201, 375, 208, 387 ActionDest: 95 Annotation: Subtype: Link Rect: 218, 363, 225, 375 ActionDest: 148 Annotation: Subtype: Link Rect: 513, 340, 525, 349 ActionDest: 242 Annotation: Subtype: Link Rect: 525, 340, 537, 349 ActionDest: 245 Annotation: Subtype: Link Rect: 400, 328, 412, 337 ActionDest: 245 Annotation: Subtype: Link Rect: 404, 300, 411, 312 ActionDest: 201 Annotation: Subtype: Link Rect: 406, 252, 418, 261 ActionDest: 245 Annotation: Subtype: Link Rect: 532, 252, 544, 261 ActionDest: 245 Annotation: Subtype: Link Rect: 544, 252, 556, 261 ActionDest: 245 Annotation: Subtype: Link Rect: 302, 240, 314, 249 ActionDest: 245 Annotation: Subtype: Link Rect: 544, 240, 556, 249 ActionDest: 242 Annotation: Subtype: Link Rect: 305, 189, 317, 199 ActionDest: 245 Annotation: Subtype: Link Rect: 320, 189, 332, 199 ActionDest: 245 Annotation: Subtype: Link Rect: 489, 189, 501, 199 ActionDest: 245 Annotation: Subtype: Link Rect: 322, 99, 329, 108 ActionDest: 239 Annotation: Subtype: Link Rect: 329, 99, 336, 108 ActionDest: 239 Annotation: Subtype: Link Rect: 337, 99, 349, 108 ActionDest: 245 Annotation: Subtype: Link Rect: 352, 99, 364, 108 ActionDest: 245 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 199, 619, 206, 631 ActionDest: 117 Annotation: Subtype: Link Rect: 410, 597, 422, 606 ActionDest: 245 Annotation: Subtype: Link Rect: 480, 572, 492, 581 ActionDest: 245 Annotation: Subtype: Link Rect: 285, 469, 297, 478 ActionDest: 242 Annotation: Subtype: Link Rect: 297, 469, 309, 478 ActionDest: 245 Annotation: Subtype: Link Rect: 312, 469, 324, 478 ActionDest: 245 Annotation: Subtype: Link Rect: 548, 456, 555, 465 ActionDest: 239 Annotation: Subtype: Link Rect: 271, 431, 283, 440 ActionDest: 242 Annotation: Subtype: Link Rect: 283, 431, 295, 440 ActionDest: 242 Annotation: Subtype: Link Rect: 519, 431, 531, 440 ActionDest: 242 Annotation: Subtype: Link Rect: 532, 431, 544, 440 ActionDest: 242 Annotation: Subtype: Link Rect: 544, 431, 556, 440 ActionDest: 245 Annotation: Subtype: Link Rect: 302, 418, 309, 428 ActionDest: 239 Annotation: Subtype: Link Rect: 309, 418, 321, 428 ActionDest: 248 Annotation: Subtype: Link Rect: 331, 406, 338, 415 ActionDest: 239 Annotation: Subtype: Link Rect: 472, 381, 483, 390 ActionDest: 242 Annotation: Subtype: Link Rect: 239, 356, 251, 365 ActionDest: 248 Annotation: Subtype: Link Rect: 402, 343, 414, 352 ActionDest: 242 Annotation: Subtype: Link Rect: 185, 293, 197, 302 ActionDest: 242 Annotation: Subtype: Link Rect: 197, 293, 209, 302 ActionDest: 248 Annotation: Subtype: Link Rect: 366, 280, 378, 289 ActionDest: 242 Annotation: Subtype: Link Rect: 372, 228, 379, 241 ActionDest: 170 Annotation: Subtype: Link Rect: 311, 165, 318, 177 ActionDest: 170 Annotation: Subtype: Link Rect: 230, 127, 237, 139 ActionDest: 170 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 345, 659, 352, 668 ActionDest: 239 Annotation: Subtype: Link Rect: 352, 659, 359, 668 ActionDest: 239 Annotation: Subtype: Link Rect: 360, 659, 372, 668 ActionDest: 242 Annotation: Subtype: Link Rect: 372, 659, 384, 668 ActionDest: 245 Annotation: Subtype: Link Rect: 398, 647, 410, 656 ActionDest: 245 Annotation: Subtype: Link Rect: 410, 647, 422, 656 ActionDest: 248 Annotation: Subtype: Link Rect: 326, 571, 338, 580 ActionDest: 245 Annotation: Subtype: Link Rect: 251, 471, 263, 480 ActionDest: 248 Annotation: Subtype: Link Rect: 266, 471, 278, 480 ActionDest: 248 Annotation: Subtype: Link Rect: 413, 458, 425, 467 ActionDest: 248 Annotation: Subtype: Link Rect: 433, 433, 445, 442 ActionDest: 245 Annotation: Subtype: Link Rect: 328, 421, 340, 430 ActionDest: 248 Annotation: Subtype: Link Rect: 388, 408, 400, 417 ActionDest: 248 Annotation: Subtype: Link Rect: 391, 345, 403, 354 ActionDest: 245 Annotation: Subtype: Link Rect: 483, 345, 495, 354 ActionDest: 248 Annotation: Subtype: Link Rect: 199, 320, 211, 329 ActionDest: 248 Annotation: Subtype: Link Rect: 214, 320, 226, 329 ActionDest: 248 Annotation: Subtype: Link Rect: 291, 308, 303, 317 ActionDest: 248 Annotation: Subtype: Link Rect: 306, 308, 318, 317 ActionDest: 248 Annotation: Subtype: Link Rect: 296, 245, 308, 254 ActionDest: 248 Annotation: Subtype: Link Rect: 352, 232, 364, 241 ActionDest: 248 Annotation: Subtype: Link Rect: 364, 232, 376, 241 ActionDest: 248 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 292, 710, 304, 719 ActionDest: 245 Annotation: Subtype: Link Rect: 424, 633, 560, 644 Annotation: Subtype: Link Rect: 166, 621, 271, 634 Annotation: Subtype: Link Rect: 104, 213, 141, 223 Annotation: Subtype: Link Rect: 277, 190, 314, 200 Annotation: Subtype: Link Rect: 519, 166, 557, 177 Annotation: Subtype: Link Rect: 59, 155, 95, 165 Annotation: Subtype: Link Rect: 488, 144, 525, 154 Annotation: Subtype: Link Rect: 95, 109, 133, 119 Annotation: Subtype: Link Rect: 139, 109, 175, 119 Annotation: Subtype: Link Rect: 351, 75, 389, 85 Annotation: Subtype: Link Rect: 395, 75, 431, 85 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 59, 710, 97, 720 Annotation: Subtype: Link Rect: 293, 676, 331, 686 Annotation: Subtype: Link Rect: 392, 652, 430, 664 Annotation: Subtype: Link Rect: 436, 652, 472, 664 Annotation: Subtype: Link Rect: 257, 618, 295, 628 Annotation: Subtype: Link Rect: 301, 618, 337, 628 Annotation: Subtype: Link Rect: 188, 584, 226, 594 Annotation: Subtype: Link Rect: 416, 560, 454, 571 Annotation: Subtype: Link Rect: 252, 525, 289, 536 Annotation: Subtype: Link Rect: 295, 525, 331, 536 Annotation: Subtype: Link Rect: 118, 492, 155, 502 Annotation: Subtype: Link Rect: 400, 456, 438, 467 Annotation: Subtype: Link Rect: 520, 433, 557, 444 Annotation: Subtype: Link Rect: 59, 423, 95, 433 Annotation: Subtype: Link Rect: 487, 399, 524, 410 Annotation: Subtype: Link Rect: 385, 364, 423, 375 Annotation: Subtype: Link Rect: 360, 330, 398, 341 Annotation: Subtype: Link Rect: 77, 296, 115, 306 Annotation: Subtype: Link Rect: 121, 296, 157, 306 Annotation: Subtype: Link Rect: 281, 272, 319, 283 Annotation: Subtype: Link Rect: 325, 272, 361, 283 Annotation: Subtype: Link Rect: 59, 238, 97, 249 Annotation: Subtype: Link Rect: 103, 238, 139, 249 Annotation: Subtype: Link Rect: 477, 215, 514, 226 Annotation: Subtype: Link Rect: 253, 181, 291, 191 Annotation: Subtype: Link Rect: 502, 146, 539, 157 Annotation: Subtype: Link Rect: 59, 112, 97, 122 Annotation: Subtype: Link Rect: 424, 88, 461, 99 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 59, 710, 97, 720 Annotation: Subtype: Link Rect: 103, 710, 139, 720 Annotation: Subtype: Link Rect: 500, 686, 537, 699 Annotation: Subtype: Link Rect: 342, 663, 379, 674 Annotation: Subtype: Link Rect: 286, 629, 323, 640 Annotation: Subtype: Link Rect: 330, 629, 366, 640 Annotation: Subtype: Link Rect: 109, 595, 146, 605 Annotation: Subtype: Link Rect: 180, 560, 217, 571 Annotation: Subtype: Link Rect: 224, 560, 260, 571 Annotation: Subtype: Link Rect: 113, 526, 150, 536 Annotation: Subtype: Link Rect: 157, 526, 193, 536 Annotation: Subtype: Link Rect: 122, 503, 160, 513 Annotation: Subtype: Link Rect: 166, 503, 202, 513 Annotation: Subtype: Link Rect: 513, 479, 551, 490 Annotation: Subtype: Link Rect: 421, 456, 459, 467 Annotation: Subtype: Link Rect: 520, 433, 557, 444 Annotation: Subtype: Link Rect: 59, 400, 97, 410 Annotation: Subtype: Link Rect: 103, 400, 139, 410 Annotation: Subtype: Link Rect: 122, 365, 159, 375 Annotation: Subtype: Link Rect: 279, 330, 317, 341 Annotation: Subtype: Link Rect: 258, 295, 296, 306 Annotation: Subtype: Link Rect: 302, 295, 338, 306 Annotation: Subtype: Link Rect: 308, 272, 346, 283 Annotation: Subtype: Link Rect: 352, 272, 388, 283 Annotation: Subtype: Link Rect: 113, 238, 150, 249 Annotation: Subtype: Link Rect: 281, 215, 318, 226 Annotation: Subtype: Link Rect: 59, 181, 97, 191 Annotation: Subtype: Link Rect: 103, 181, 139, 191 Annotation: Subtype: Link Rect: 59, 146, 97, 157 Annotation: Subtype: Link Rect: 176, 112, 213, 122 Annotation: Subtype: Link Rect: 219, 112, 255, 122 Annotation: Subtype: Link Rect: 113, 77, 150, 88 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 223, 709, 261, 720 Annotation: Subtype: Link Rect: 86, 676, 123, 686 Annotation: Subtype: Link Rect: 130, 676, 166, 686 Annotation: Subtype: Link Rect: 86, 641, 124, 651 Annotation: Subtype: Link Rect: 130, 641, 166, 651 Annotation: Subtype: Link Rect: 170, 607, 208, 617 Annotation: Subtype: Link Rect: 498, 583, 536, 595 Annotation: Subtype: Link Rect: 324, 548, 361, 559 Annotation: Subtype: Link Rect: 324, 526, 362, 536 Annotation: Subtype: Link Rect: 368, 526, 404, 536 Annotation: Subtype: Link Rect: 500, 502, 537, 513 Annotation: Subtype: Link Rect: 496, 479, 534, 492 Annotation: Subtype: Link Rect: 343, 456, 380, 467 Annotation: Subtype: Link Rect: 59, 423, 97, 433 Annotation: Subtype: Link Rect: 519, 399, 557, 410 Annotation: Subtype: Link Rect: 59, 388, 95, 398 Annotation: Subtype: Link Rect: 207, 354, 245, 364 Annotation: Subtype: Link Rect: 333, 318, 370, 329 Annotation: Subtype: Link Rect: 412, 295, 449, 306 Annotation: Subtype: Link Rect: 438, 272, 475, 283 Annotation: Subtype: Link Rect: 481, 272, 517, 283 Annotation: Subtype: Link Rect: 408, 238, 445, 249 Annotation: Subtype: Link Rect: 407, 203, 445, 214 Annotation: Subtype: Link Rect: 451, 203, 487, 214 Annotation: Subtype: Link Rect: 506, 180, 544, 191 Checksum: f973b4a1 Type: CRC32 Checksum: eb5be901c744b0ed94c4192f79991233 Type: MD5 Checksum: 2e70b7c5e5932de156f95309538112274248d087 Type: SHA-1