9/21/10 1 EC Coupling in Heart Calcium Cycle in Cardiac Muscle!9/21/10 2 Calcium Transients.!9/21/10 3 Cardiac signaling Cell Vm Iion ΔL/L [Ca2+]i Ipropagation Cell #2 #1 Some Charactersistics of EC Coupling • Sidney Ringer clearly recognized that extra cellular Ca is required for contraction in heart. • In heart cells contraction can be graded rather than all or none. • To produce graded contractions EC coupling in heart must possess mechanisms that eliminate positive feedback • Mechanisms and theories of EC coupling must accommodate these facts.9/21/10 4 Ca2+ sparks and the Ca2+ transient 1 3 F/F0 200 pA 100 ms + 1 µM nifedipine 200 ms -60 +10 mV Control +10 mV From Fawcett and Mc Nutt , 19699/21/10 5 Ca Ca Ca Ca Ca 3Na Ca ATP RyR MF C!a!C!a!3!N!a!Terminal Cisternae!SR Release Channel (RyR)!Ca Induced Ca Release:Trigger Hypothesis!L type Ca Channel!9/21/10 6 Ca!RyRs Regeneration Trigger Current Cell Membrane SR Ca!Ca!Ca PMT!Light Source!Laser!Objective!Specimen!Focus!Beam Splitter!Detector!Aperture!Source Aperture!9/21/10 7 Scan 2!Scan 1!T-! Tubule!Emission Line 1!Emission Line 2!Local Ca!transient!Stacked Lines!t!x!Cell loaded wi!th Fluo 3!92 µm!58 ms!Line Scan Plot of Evoked Mouse!Sparks 255!0!9/21/10 8 SR Sarcolemma Ca2+ Ca2+ trigger RyR LCC Preliminary Ideas on Couplons SR Sarcolemma Ca2+ Ca2+ trigger RyR LCC9/21/10 9 Small Junction:Ca can Rise to 30µM and Gate Release Channels and Produce local Ca release events t Tubule SR Terminals Numerous Junctions Spaced to Ensure Adjacent Junctions not Activated by Ca Release Ca Local Control Is a Single RyR Responsible for Producing a Spark • It is important to understand two aspect of the structure of junctional regions • The first is the number of RyRs responsible for generating a spark • The second is the number of L-type Ca channels required to trigger a spark9/21/10 10 Single Channel Sparks • Open time frequency histogram exhibits an exponential distribution • Spark amplitude ∝ ∫Ca fluxryr.dt • Therefore we expect spark amplitudes to distribute exponentially if they are produced by a single channel9/21/10 11 The Structure of the Junction Although is appears that a cluster of RyRs contribute to the formation of a spark, it is not clear how many L-type Ca2+ channels are required to ensure that a spark is triggered. We investigated this by examining both the properties of Ca2+ sparks evoked by action potentials and single channel activity in rabbit ventricular myocytes.9/21/10 12 Sparks and T-tubule locations 50 ms Sulforho- damine B 5 µm Sulforhodamine B 10 µm Sulforhodamine B occupied T-tubules fluo-3 recorded simultaneously 1.0 2.0 1.5 #5 #8 #9 #10 #11 F/F0 images Sparks were evoked during action potentials. They were abolished after treatment with thapsigargin. T-tubule locations Average T-tubular spacing = 1.7 µm. Sparks appeared coinciding with T-tubules. Average of 23 F/F0 images Sparks produced by sequential stimuli 40 ms 1.0 2.0 1.5 F/F0 F/F0 10 µm Vm 0 -100 mV #1 #50 50 series of action potentials and F/F0 images Sparks observed at a fixed location and confocal plane appeared at the beginning of nearly every action potential. The amplitude of the sparks was variable among sites, but approximately constant at a given site.9/21/10 13 Properties of signal-averaged spark Spark size = 1.8 µm (at half maximum width) Spark profiles and action potential 10 ms 0-100 mV F/F0 1!1.5!2!stimuli 0.0 µm 0.3 µm 0.6 µm 0.9 µm 1.2 µm distance from center Averaged single spark 1.0 2.0 1.5 F/F0 1 µm 10 ms stimuli 10% of the peak of every spark occurred with latency of 2 - 6 ms from the peak of every action potential. Probability of null sweeps ♦ We detected all open events in 1000 sweeps during the period 2 - 6 ms after the clamp pulses. ♦ Open events are detected at half-amplitude threshold. ♦ From this, we calculated the probability of null sweeps at +10 mV and +50 mV. #420 #292 #102 2 6 20 ms Pnull+10mV = 0.58 Pnull+50mV = 0.17 #151 #11 #389 2 6 20 ms9/21/10 14 Summary - Ca2+ sparks • The properties of sparks in rabbit ventricular myocytes are similar to those in other species. • Sparks at fixed sites during a series of action potentials occurred at the beginning of nearly every action potential, i.e., they appeared with a probability ~ 100%. Close examination of spark probability revealed a small proportion of spark failures, i.e., 0.3%. • Sparks occur in a limited interval 2 - 6 ms after the action potential peak. SR Sarcolemma Ca2+ Ca2+ trigger RyR cluster of as many as 150 channels LCC LCC LCC LCC LCC LCC LCC LCC LCC LCC cluster of possibly 30+