Doctors Revision

Doctors Revision

TOAD HEART IN SITU & PROPERTIES OF CARDIAC MUSCLE

EXPERIMENT 1: TOAD HEART IN SITU AND PROPERTIES OF CARDIAC MUSCLE

Objectives

  • Describe the method of isolation of the toad heart.
  • Determine the effect of temperature on cardiac muscle.
  • List the effect of different ions and drugs on the isolated heart muscle.
  • Explain the mechanism of action of drugs and ions on the cardiac muscle.
  • List the properties of the cardiac muscle.
  • Elaborate the physiological basis of different properties of the cardiac muscle.

Introduction

The naturally beating toad heart is first observed in situ with its apex connected to a writing lever for recording the sequence of events during contraction. The heart rate is altered by changing the temperature of the bathing fluid. Electrical stimuli are applied between beats to illustrate properties of the conducting system of the heart.

Once the conducting system has been inactivated by crushing, cardiac muscle can be studied as a muscle preparation. Cardiac muscle has a different stimulus-response relationship from skeletal muscle, and it shows refractoriness to a second stimulus at some stimulus intervals.

Apparatus

Kymograph

A motor-driven rotating drum that operates at four different speeds, equipped with a clutch mechanism.

The drum carries smoked paper that is written on by various levers.

Note: Traces must be fully labeled including student names before being shelled.

Induction Coil

Provides either single stimuli or repetitive stimuli.

Note: Relative stimulus strength must always be recorded as the distance in centimeters between primary and secondary coils.

Preparation


A. Dissection

  1. Use a pithed toad (brain and spinal cord destroyed) placed on its back on a cork board.
  2. Pin through the web of each foot and the lower jaw.
  3. Expose the xiphisternum (cartilaginous extension of the sternum).
  4. Make a transverse incision through the abdominal wall below the xiphisternum.
  5. Cut through both sides of the sternum and pectoral girdle.
  6. Remove anterior thoracic wall.
CRITICAL:

Frequently irrigate tissues with physiological saline to prevent dessication (drying out).

  1. Display thoracic contents by repinning front feet wider apart.
  2. Carefully incise the pericardium laterally and reflect it back.
  3. Observe heart action and identify successive contractions of the sinus venosus, atria, ventricles, and truncus arteriosus.

B. Mounting for Recording

  1. Tie silk thread to a fine hook and pass through the ventricle tip without puncturing tissue.
  2. Gently lift heart and cut the transverse pericardial ligament (between atria and venous side).
  3. Transfer toad to recording stand bath.
  4. Anchor heart base with pin through connective tissue near the aorta.
  5. Keep heart moist with Ringer's solution but do not fill the bath yet.
WARNING:

Skin secretions are toxic—prevent bath fluid contamination.

  1. Tie silk thread to the hole nearest the heart lever pivot (must be precisely vertical).
  2. Adjust lever vertically so it's horizontal when heart is relaxed and thread is just taut.
  3. Adjust kymograph for maximum friction.
  4. Adjust lever spring for 1-2 cm amplitude tracing.

EXPERIMENTAL PROCEDURES


A. Heart Beat & Temperature Effects

1. Baseline Recording
  • Speed: Moderate (25 mm/sec).
  • Observation: Make a short record. Relate lever movements to actual heart chambers—identify up to four contractile events.
2. Temperature Effects (General)
  • Speed: Slow (2.5 mm/sec).
  • Temps: Bathe heart with saline at approx 0°C, 10°C, and 20°C.
  • Note: Ensure pipette is cooled/heated by solution. Measure temperature accurately. Use signal marker and clock for time traces.

Alternate Temperature Procedure

  1. Label beakers: 0°C, 10°C, 20°C, 30°C, 40°C.
  2. Add 3 mL frog Ringer's to each.
  3. Immerse muscle at 0°C, record twitch.
  4. Replace with 20°C and 30°C, wait 30 seconds, record.
  5. Replace with 10°C, wait 1 minute, record.
  6. Replace with 40°C, record irregular twitches.
  7. Analysis: Draw lines from curve summits to baseline. Record graph heights (cm) and durations.

Data Table 1: Heart Rate vs Temperature

Temperature (°C) Heart Rate (beats/min) Observations
0
10
20
30
40

B. Refractory Period of Conducting System

  • 1 Place Electrodes: One against auricles, other against ventricle. Note: Must not impede movement.
  • 2 Settings: Set signal marker in primary circuit for single break stimuli. Run drum at moderate speed (25 mm/sec).
  • 3 Stimulus Strength: Move secondary coil to produce supra-maximal stimuli (8-10 cm on scale).
  • 4 Procedure: Apply single stimuli at various times during the cardiac cycle (systole and diastole).
    • Measurement Required:

    Determine refractory period duration and maximum "compensatory pause".

    C. Mechanical Block of Conduction (Stannius Ligatures)

    Preparation:

    Pass moistened silk thread between aortae and veins, tie loosely. Record at slow speed (2.5 mm/sec).

    First Ligature (Sinus-Atrial)

    Tighten ligature across the sinus venosus-atrial junction (white crescent).

    Effect: Crushes conducting tissues to auricles; sinus continues beating alone while the rest of the heart may stop temporarily.

    Second Ligature (Atrio-Ventricular)

    Tie between atrium and ventricle across the atrioventricular bundle.

    Effect: Isolates the ventricles from the atria.
    Measurement Required:

    Determine the inherent rates of the auricles and ventricles separately after isolation.

    D. PROPERTIES OF CARDIAC MUSCLE

    1. Stimulus-Response Relationship

    1. Set secondary coil at maximum distance from primary coil.
    2. Apply single break stimuli to ventricle (both electrodes) at ~15-second intervals.
    3. Between stimuli, turn drum ~1 cm by hand to separate traces.
    4. Successively increase stimulus strength (move coils closer) until ventricle responds.
    5. Record cm position of secondary coil for each response.
    6. Find sub-threshold stimulus, then switch to repetitive stimulation.
    7. Observe response to brief repetitive stimulation.

    2. Refractory Period of Directly Stimulated Muscle

    1. Reconnect for single stimuli. Set supra-threshold stimulus strength.
    2. Run drum at moderate speed (25 mm/sec).
    3. Apply paired stimuli by two quick taps of telegraph key (< 1 second intervals).
    4. Measurement: Determine the maximum interval without a second contraction. This represents the refractory period.
    5. Repeat with increased stimulus strength (refractory period should shorten).
    6. Apply brief repetitive supra-threshold stimuli—compare response to single stimulus.

    E. EFFECT OF IONS ON HEART IN SITU

    Ion Effects Overview:
    • Isotonic NaCl: Rhythm disappears, beating ceases.
    • CaCl₂: Heart beats briefly, then stops in systole (contraction).
    • KCl: Heart stops in diastole (relaxation).
    • Ringer's solution (all three ions): Beating continues indefinitely.
    Ringer's Solution Composition: NaCl: 0.9 g
    CaCl₂: 0.024 g
    KCl: 0.042 g
    NaHCO₃: 0.02 g
    Distilled water to 100 mL

    Procedure

    1. Bathe heart with Ringer's until baseline rate established.
    2. Prepare NaCl, KCl, CaCl₂ at 3× concentration.
    3. Apply 5 mL of each solution onto heart.
    4. Application Order: NaCl → CaCl₂ → KCl.
    Critical:

    Wash thoroughly with Ringer's between each application. Ensure heart returns to baseline rate and rhythm before adding the next solution.

    Data Table 2: Ion Effects

    Substance Heart Rate / Observation
    Ringer's Solution
    Sodium Chloride
    Calcium Chloride
    Potassium Chloride

    F. EFFECT OF DRUGS ON HEART IN SITU

    Apply adrenaline and acetylcholine using the same procedure as ions (apply, observe, wash, recover).

    Data Table 3: Drug Effects

    Drug Heart Rate / Observation
    Adrenaline
    Acetylcholine

    ANALYSIS OF RESULTS

    A. Data Tables

    • Temperature Effects: Columns for measured temperature, logarithm of temperature, and heart rate (beats/min).
    • Stimulus-Response: Columns for applied stimulus (secondary coil position in cm) and muscle contraction (mm deflection).

    B. Graphs to Plot

    • HR vs Log Temp: Heart rate (ordinate/y-axis) against log of temperature (abscissa/x-axis).
    • Contraction vs Stimulus: Contraction (mm, ordinate) against stimulus strength (cm, abscissa). Note: Weakest stimulus at origin; abscissa scale decreases left to right.

    C. Calculations (Q₁₀)

    Calculate the temperature coefficient (Q₁₀):

    Q₁₀ = (Heart rate at higher temp) ÷ (Heart rate at lower temp)

    (For a 10°C rise)

    Compare Q₁₀ values for different temperature ranges (e.g., 0-10°C vs 10-20°C) and explain similarities/differences.

    QUESTIONS

    1. Temperature Analysis

    How did temperature (heat and cold) change the heart rate from baseline? Explain the physiological mechanism.

    2. Chemical Mechanisms

    Describe the effect that you would expect each chemical (Ions & Drugs) used to have on heart rate and amplitude, and explain your reasoning based on cardiac physiology.

    Physiology Steeplechase: Toad Heart In Situ

    Physiology Steeplechase

    Toad Heart & Cardiac Muscle Properties

    What to identify:

    • Apparatus: Identify the Kymograph and setup.
    • Tracings: Interpret the effect of Temperature, Ions, and Drugs on the graph.
    • Mechanisms: Explain why the curve changed (e.g., Systolic vs Diastolic arrest).
    Shopping Basket