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Objectives: by the end of this combined exercise you should be able to:
|10.00 hrs||Seminar and small group work||Worsley Room 6.55|
|12.00 hrs||Lunch break|
|13.00 hrs||Practical work||Irene Manton cluster|
|16.00 hrs||End of practical||Show your results record and computer print out to your demonstrator|
Webpages: We recommend that you visit the neighbouring sections on muscle biochemistry and computer data acquisition. You could also visit the bioenergetics pages on this website which might be helpful for BIOC2300 and BIOC2120 exam revision.
Assessment: A written report is not required for this practical. Course work marks are obtained for attendance and completion of the results proforma at the end of this practical schedule. You must show your completed results record and your final computer print out to a demonstrator before leaving the lab. Questions on this practical may appear on the end-of-module examination.
This seminar is divided into 4 parts.
Cardiac muscle cells are striated, nucleated, electrically excited cells. Their function is to contract and relax rhythmically. They are joined by special structures called intercalated discs. The intercalated discs contain gap junctions which allow action potentials to spread rapidly from one cell to the next so that the contraction of the cells is synchronised.
The ionic composition of the cytoplasm is tightly controlled in all cells. The predominant mechanisms involved in this control are represented in Figure 1 below. The sodium pump, in conjunction with secondary ionic exchangers, maintains steep concentration gradients for sodium, potassium and calcium across the plasma membrane. The passive permeability for potassium ions is greater than the permeability to sodium and calcium. Under resting conditions calcium ions and sodium ions tend to diffuse down their concentration gradients into the cell, but are unable to reach equilibrium. Potassium ions, in contrast, are close to equilibrium, and the tendency of potassium ions to leave the cells down the potassium concentration gradient is exactly balanced by the electrical membrane potential operating in the opposite direction across the plasmalemma.
The sodium pump (Na+/K+-ATPase) maintains the gradients for Na+ and K+ by expelling three Na+ ions from the cell and taking in two K+ ions for every molecule of ATP consumed. Although this process provides the ultimate driving force for many of the subsequent ionic movements, it is NOT the immediate cause of the membrane potential. We know this because the resting membrane potential and the ability to generate repeated action potentials persists for some time after the sodium pump has been completely inhibited. How exactly does the resting potential arise?
The cytoplasmic Ca2+ concentration is also controlled by an ATP-dependent pump protein in the membrane of the sarcoplasmic reticulum which temporarily sequesters Ca2+ into the lumen of this calcium storing organelle.
Under normal conditions the main event which disturbs the ionic gradient across the plasma membrane is the action potential which is recorded as a rapid depolarisation and subsequent slower repolarisation of the transmembrane potential (Figure 1). Separate voltage sensitive channels for Na+, K+ and Ca2+ are present in the plasma membrane which transiently open and increase the passage of these ions down their electrochemical gradients. The Na+ channels open first and then rapidly inactivate. At potentials positive to -40 mV the Ca2+ channels open but inactivate more slowly than the Na+ channels. Subsequent opening of voltage sensitive K+ channels allows the efflux of K+ and the depolarisation induced by the inflow of Na+ and Ca2+ is countered. As K+ efflux increases as the membrane potential becomes more negative, the cardiac cell action potential contains a plateau phase followed by a rapid repolarisation phase. A second class of K+ channels is also present in the plasma membrane These remains closed (inactive) in the presence of ATP but if ATP levels fall and these channels open, efflux of K+ leads to the cell becoming hyperpolarized and inactive.
The contraction and relaxation of these cells is effected by cyclic association and dissociation of actin and myosin. This cycle is regulated by Ca2+, the troponin complex and tropomyosin. The troponin complex consists of three subunits: troponin C, troponin I and troponin T. Troponin C binds Ca2+, troponin I binds to actin and troponin T binds to tropomyosin. When the cytoplasmic Ca2+ concentration is raised Ca2+ binds to troponin C inducing troponin C to change shape and to bind more strongly to troponin I which weakens the bonds between troponin I and actin. This alters the interaction between troponin T and tropomyosin, the position of tropomyosin and reveals the sites which allow myosin to bind to actin. The 'cross-bridge' cycle is then initiated and if repeated at many points along the muscle filament contraction occurs. A separate thick filament regulatory system is also present, but plays a subordinate role in striated muscles.
Catecholamines such as adrenaline increase the force of cardiac contraction by (among other things) phosphorylating the plasmalemma calcium channels and increasing the entry of calcium ions to the cytosol during the plateau phase of the action potential.
The force of cardiac contraction can be reduced by drugs such as verapamil and nifedipine, which inhibit the initial entry of calcium ions through the plasmalemma. These drugs also promote relaxation of vascular smooth muscle through a similar mechanism. This intervention may be useful in patients suffering from hypertension and ischaemic heart disease.
To bring about relaxation Ca2+ must be removed from the cytosol and ATP must be present. ATP is required both for pumping most of the Ca2+ back into the sarcoplasmic reticulum, and (indirectly) for removing surplus Ca2+ from the cells [see below]. ATP is also required for relaxation of the actin-myosin cross bridges within the myofilaments.
It is apparent from the above that the force developed by cardiac muscle cells depends partly on the cytoplasmic Ca2+ concentration. Other factors can also influence the force of contraction. Cellular acidosis (increased cytoplasmic H+ concentration) decreases the sensitivity of the contractile elements to Ca2+ so that the force developed at a given cytoplasmic Ca2+ concentration is reduced. Inorganic phosphate also influences contractile activity by reducing maximum developed force as well as reducing the sensitivity to Ca2+ so that 20 mM inorganic phosphate (a concentration achieved during ischaemia) reduces the force to 20% of that developed under normal conditions.
Many cells use the steep Na+ gradient to drive an Na+/H+ exchange protein that extrudes H+ from the cell. This cannot be the only mechanism for intracellular pH control, because cell interiors are normally slightly acidic when compared with the external medium. In cardiac muscle, BUT NOT IN OTHER TISSUES, the sodium gradient is also utilised by the Na+-Ca2+ exchange protein to extrude Ca2+ from the cells. This is the basis for the inotropic action of the cardiac glycoside, digitalis, and also ouabain. Can you explain in detail how these compounds work?
Visit the muscle biochemistry pages for a more detailed account of the structure and function of cardiac muscle.
Draw your own schematic of the complete depolarisation/repolarisation, activation-contraction cycle
You are provided with raw data from an experiment with a new cardiac drug. During your session in the Irene Manton Cluster:
The strength of contraction of cardiac muscle cells can be altered either by changing the amount of Ca2+ released into the cytoplasm or by altering the response of the contractile proteins to the available Ca2+. Inotropic agents (drugs which increase the force of contraction) may act by either of these mechanisms.
A new compound synthesised by company X had been shown in preliminary tests to have inotropic activity. But its mechanism of action was unknown. An experiment was carried out to determine whether it was increasing the Ca2+ available to the contractile mechanism or whether it was sensitising the contractile mechanism to Ca2+.
A small piece of heart muscle (diameter 0.6 mm) was mounted in a muscle bath between a fixed hook and a force transducer. The muscle was continuously superfused with a physiological salt solution at 30°C, and was electrically stimulated to contract once every 3 sec. A protein called aequorin was microinjected into several superficial cells. As aequorin emits light when it binds Ca2+ the cytoplasmic [Ca2+ ] can be recorded by measuring the light emitted by the intracellular aequorin. [This experiment was done a few years ago. Nowadays the aequorin gene would be introduced directly into the mammalian genome, and its expression directed towards the appropriate tissue compartment by including the correct promoters and protein targeting sequences.]
The output from the force transducer was 5 volts for every 2.5 g wt developed by the muscle (calibrated by hanging weights on the force transducer) . The signal from the force transducer was digitised using a 12 bit analogue to digital (A/D) converter with a full scale of -5 V to +5 V. The digitised signal was then recorded, averaged and stored using a computer. A sampling speed of 1000 Hz was used (i.e. a data point was collected every msec), and recordings were made for 1 sec (i.e. each record contained 1000 points). The muscle was electrically stimulated 100 msec after the beginning of each record.
A photo-multiplier was used to detect the emitted light. The output current from the photo-multiplier was converted to a voltage and then amplified so that each 100 nA produced by the detector corresponded to 5 V. This signal was also digitised and stored on the computer as described above for the force of contraction.
Force of contraction data and aequorin light data were collected when the tissue was stimulated under control conditions (file 15mar00.xls). Data was then collected in the presence of 10 uM of the compound (file 15mar04.xls). The data cannot be interpreted in this raw form. It has to be first manipulated and visualised by plotting. You are to carry out this. Guidelines are given below.
Note: Instructions are in boldface type whereas information is given in normal type.
Click here to download 15mar00.xls to your local disk.
Click here to download 15mar04.xls to your local disk.
Ignore any warning messages from Netscape about virus infection: these files are from a "trusted" internal source and should be clean. SAVE them to your local disk drive. You could alternatively use Windows Explorer to copy the files from n:\windept\bmb\bioc2110 either to your network drive or to a floppy disc. If you use this second method, remove the "read only" attribute from these files using the "File: Properties" command in Explorer, or you will not be able to update them.
You should be using version 5 of Excel (from Office 97) for this practical. This edition is more straightforward than its predecessors. The printed version of these instructions contains a cryptic reference to the "Gallery/Combination" command in section 6. This was used for a previous version of Excel and should be ignored. Use the Chart Wizard and Format data series menus instead.
The file consists of two columns of data, column A contains the aequorin light data and column B contains the force data. Each column contains 1000 points. The rows therefore contain digitised signals collected at 1 msec intervals: increasing row numbers represent increasing time, while the numbers in the rows represent the signals at particular time points. The raw data is "tab delimited" text. You are strongly advised to save it in the "proper" Excel spreadsheet format.
Start by placing a 1 in cell A2 and the formula =A2+1 in cell A3. Reselect cell A3 before scrolling to the last row which contains any data, without clicking elsewhere in the chart. FIRST HOLD DOWN THE SHIFT KEY and then highlight all the intervening cells in the first column by clicking in the bottom cell. Release the shift key and use the Edit: Fill Down command to fill in the remaining 998 entries automatically.
Detailed instructions have deliberately not been given. Ask your demonstrator if you get really stuck. You are to investigate the functions within EXCEL to reproduce the style shown in the representative figure in the module manual.
Transducer calibration is described in the Experimental Section. 2047 = 0V and 4095 = +5V. Visit the data acquisition pages for a detailed description of the data conversion and calculations. Keep the Time column to the left and insert calculated data into new columns.
The Light and Force data are now in different units so click on each curve in turn, and use the "Format data series: Axis" command to assign it to either the primary (left hand) vertical axis, or the secondary right hand scale.
All this material should fit easily onto one page.
If you have comments, queries or suggestions, email me at J.A.Illingworth@leeds.ac.uk