1. Donna H. Korzick, Ph.D. Noll Physiological Research Center and The Department of Kinesiology Regulation of Cardiac EC-Coupling: A Cellular Update Experimental Biology 2003: APS Refresher Course San Diego, CA

2. Muscle Physiology: From Cellular to Integrative Regulation of Cardiac Performance I.Microdomains and Local Control of EC Coupling Transarcolemmal Ca 2+ fluxes Ca 2+ sparks Ca 2+ release mechanisms II.What’s “Relatively” New in Adrenergic Signaling? Are  1 -ARs the only receptors responsible for changes in contractility? GPCR’s and beyond III.What Should Our Students Know About Intracellular Signaling? Protein scaffolds Receptor Desensitization IV.“Relatively” New Ideas Nitric Oxide as a regulator of cardiac performance

3. Muscle Physiology: From Cellular to Integrative Regulation of Cardiac Performance I.If you only have 6-10 lectures, what do you teach? APS CV Objectives II.Is there a perfect textbook? No Supplement with good review articles

4. VO 2 = Cardiac Output x a-vO 2 diff Cardiac Output = Heart Rate (HR) x Stroke Volume (SV) A. Regulation of pacemaker activity (HR) B. Regulation of myocardial performance (SV) C. Major Points 1. principle control of HR is by the ANS (extrinsic) 2. both intrinsic and extrinsic mechanisms regulate SV 3. contractility is defined as cardiac performance independent of changes in preload and afterload

5. Efflux Influx Cooperative binding to myofilaments (Ca + sensitivity) Typical twitch – contractile force reaches ~45% of max (requires 70  mol/L cytosol = ~600 nmol/L [Ca] i Bers, Circ Res: 87:275-281,2000 VGCC’s NCX SR Ca 2+ -ATPase NCX SL Ca 2+ -ATPase Mitochondrial Ca 2+ Uniporter

6. What underlies the [Ca 2+ ] I ? The elementary event of SR Ca 2+ release in cardiac muscle is the Ca 2+ spark “Local Control of EC-Coupling” The [Ca 2+ ] i arises as Ca 2+ sparks sum Point: Mechanisms that alter cytosolic Ca 2+ or myofilament Ca 2+ sensitivity alter LV developed force Central Dogma for Cardiac EC-Coupling Ca 2+ -Induced Ca 2+ Release Small Ca 2+ increases in the vicinity of the SR lead to much larger Ca 2+ release from the SR Electrical excitation at the SL membrane activates VGCCs (DHPR) Influx of Ca 2+ via the [Ca 2+ ] I [Ca 2+ ] I activates Ca 2+ release channels on the SR (RYR) Contractile element activation

7. “Normal EC coupling involves a well-ordered and stereotyped sequence of events” Ca 2+ Sparks : *represent Ca 2+ passing thru RYRs *represent small local Ca 2+ release events *sparks can be evoked by depolarization and AP’s Guatimosim et al, J Mol Cell Cardiol, 34: 941-950, 2002 E 0.5 s

8. Why Ca 2+ Sparks? 1.Summation of sparks provides the microscopic basis of the [Ca2+] i 2.Provides an explanation for graded contractions which are modulated by local control 3.Provides insight into microdomains and where they occur 4.Provides insight into defects into pathological changes in EC Coupling Calcium waves and arrhythmias

9. RYR2 = Ca 2+ release channel Phosphorylated by PKA, ? PKC, ? PKG, ? CamKII scaffolding protein that localizes numerous key regulatory proteins to the junctional complex RyR2 as a Ca 2+ Release Channel Bers, Nature, 415: 198-205, 2002

10. Marks, J Mol Cell Cardiol, 33: 615-624, 2001 RyR2: -tetrameric channel -4 ~565,000 dalton subunits ->2.3 million daltons -FKBP12.6 -PKA -AKAPs -PP1 (spinophilin) -PP2A (PR130) RyR2 as a Macromolecular Signaling Complex Point: Local control of RyR2 function by macromolecular signaling complexes allow for a graded physiological response to stress

11. Functions of FKBP12.6: 1.Stabilize the closed state of the RYR to prevent Ca 2+ leak 2.“Coupled Gating” 3.Regulate RyR2 sensitivity to Ca 2+ activation Bers, Nature, 415: 198-205, 2002 Role of FKBP12.6 on RyR2 Regulation

12. Physiologic Regulation of the Inotropic State 1. Adrenergic Stimulation 2. Myocardial Performance Frank-Starling Effect Force-Frequency Relation 3. Adrenergic Regulation 4. Modified from Ross et al, Circulation 1995; 92: 2327-2332 5. Vascular Function

13. Figure 24-18 Contraction and Relaxation are Enhanced by SNS Stimulation Figure 24-24 Berne and Levy

14. Signal  Receptor  Coupling Protein Response Second Messengers  Response G Proteins Gs Gs/Gi Gq  1 -ARs  2 -ARs  1 -ARs cAMP (PKA) PDE/PP DAG (PKC) IP 3 Signal Transduction of Myocardial Performance

15. Rockman et al, Nature, 415: 206-212, 2002 G-Protein-Coupled Receptor Signaling

16. Sympathetic Influences on Myocardial Contraction: NE

17. (PKA) 1.2.3. Point:  1 -adrenergic stimulation increases both cardiac inotropy and lusitropy. Point: Mechanisms that alter cytosolic Ca 2+ alter LV developed force Reducing Na + gradient (  Na + i /  Na + o ) 4. Berne and Levy

18. Bers, Nature, 415: 198-205, 2002 RyR2 is also a target of PKA phosphorylation Bottom line: increase P o of RyR2

19. Dual Coupling of  2 -ARs to G s and G i

20. Inhibits I to (so what?)  Contractility Gq-coupled GPRCs confer positive inotropic effects and myocardial cell growth

21. Important Seven-Transmembrane-Spanning Receptors Modified from Rockman et al, Nature, 415: 206-212, 2002

22. Chemical Control of Myocardial Performance 1.Epi (adrenal medulla)/ Norepi:  1,  2,  1 - ARs 2.Ang II: positive inotrope (G q, IP 3 /PKC pathway) 3.Opioids: positive inotrope (G q, IP 3 /PKC pathway) 4.Endothelin I: positive inotrope (G q, IP 3 /PKC pathway) 5.Thyroid Hormone: positive inotrope/chronotrope (genomic and nongenomic effects) 6.Insulin: positive inotrope (PKC, PI3K) 7.Glucagon: positive inotrope/chronotrope (AC)

23. Rockman et al, Nature, 415: 206-212, 2002 Receptor Desensitization: Agonist-Induced Phosphorylation of  ARs by GRK2 GRK2 =  Adrenergic Receptor Kinase 1 (  ARK1)  1/2 -AR

24. Endothelial Cell Smooth Muscle Heart “Relatively” New Ideas: Nitric Oxide as a Mediator of Cardiac Performance

25. NO as a positive inotrope NO as a negative inotrope NO modulates  -AR effects on contractile performance eNOS production by cardiac myocytes in response to “stretch” Machinery for endogenous NO production resides in cardiac myocytes May subserve SR Ca 2+ release functions PI3K-dependent phosphorylation of Akt and eNOS  increased NO production eNOS as a regulator of mitochondrial respiration iNOS as protective nNOS as a regulator of SERCA May subserve Ca 2+ reuptake mechanisms “Relatively” New Ideas: Nitric Oxide as a Mediator of Cardiac Performance

26. Thank You