Cardiovascular Function In Rana pipiensOverviewEquipment RequiredSolutionsEquipment SetupStart the SoftwareThe DissectionHints:Procedure Exercise 1: The Resting Heart RateProcedureExercise 2: The Effects of Neurotransmitter on Contractile Force and FrequencyProcedureNorepinephrine (or Epinephrine)Exercise 3: The Effect of Cold Temperature on the HeartExercise 4: Identification of an Unknown Pharmacological AgentData AnalysisExperiments 2 & 3:Thought QuestionsCardiovascular Function In Rana pipiensOverviewThe rate and force of ventricular contraction (contraction is often referred to as “systole”) are controlled by various intrinsic and extrinsic factors acting on the heart at a given instant. A vertebrate heart is myogenic, or able to generate its own contractions and maintain the pace of the heartbeat without input from other parts of the body, including the nervous system. Despite this ability, the heart rate is often controlled, at least partially, by the autonomic nervous system (ANS) and/or endocrine system, which discharge chemicals that alter both the rate and force of ventricular contraction. The Autonomic Nervous System (ANS) is composed of two antagonistic branches that control many of the organs and tissues of the body. These two branches, the sympathetic and parasympathetic branches, dominate at different times and under differing conditions. The sympathetic division of the ANS, often referred to as the “fight or flight” system, reigns during times of perceived danger, anxiety, fear or excitement. This division tends to speed up the heart rate (positive chronotropy) when its nerves discharge norepinephrine (NE) onto Beta 1 adrenergicreceptors on the heart. The parasympathetic branch of the ANS, often referred to as the “rest or digest” system, reigns during times of relaxation. This division tends to slow the rate of contraction (negative chronotrophy) when its nerves discharge acetylcholine (Ach) onto muscarinic cholinergic receptors on the heart. The overall heart rate at any given moment tends to reflect the extent to which either branch of the ANS is actively secreting its product(s) onto the heart.The endocrine system can also modulate heart rate and force of contraction, primarily through therelease of Epinephrine (Epi). Unlike norepinephrine and acetylcholine, epinephrine is a neurohormone. It is stored and released by a gland (the adrenal gland) and travels through the blood stream to its target tissues. One such target tissue is the heart. Epinephrine, like norepinephrine, binds to B1 adrenergic receptors and brings about a similar physiological response (+ chronotrophy). External conditions, when imposed on an animal, can induce changes in heart rate. For example,infusion of the heart with a pharmacological agent can either enhance or inhibit the activity of one or both branches of the ANS. Some chemicals, generally categorized as “agonists”, enhance the activity of a neurotransmitter or mimic the action of that neurotransmitter at its receptor site. The physiological response to an agonist is often indistinguishable from the neurotransmitter that it mimics or assists. Other chemicals act by blocking or retarding receptor sites to which a neurotransmitter binds. These chemicals are known as “antagonists” and, depending upon the receptor upon which they act, may halt or even reverse the physiological effect of a neurotransmitter. The force and rate of ventricular contraction are tightly linked in some organisms, such that force is often influenced by the rate (but NOT visa versa). Starling’s Law of the Heart illustrates an attempt to explain the link between heart rate and force of contraction. This law states that the amount of stretch of the myocardium (muscle cells of the heart) determines, to a great extent, the amount of force produced during contraction. Specifically, the greater the distention of the ventricle at the time of contraction, the greater the amount of force developed by the muscle and the greater the volume of blood ejected by way of contraction. The amount of blood ejected (in mL) per contraction is known as the stroke volume (SV). Since heart rate determines the amountof time the ventricle has to fill with blood between contractions, heart rate tends to be inverselyrelated to myocardial stretch and the force of contraction. Following this logic, it is fair to assume that as heart rate increases (and ventricular filling time decreases), the level of stretch and the force produced during contraction decrease. So does the stroke volume. This law has been illustrated in a number of animal models, including humans. Despite the validity of Starling’s Law, there are some important exceptions that should be addressed. Certain substances (both endogenous and exogenous) have been shown to defy Starling’s Law and its predictions. One such example is norepinephrine, the main neurotransmitter of the sympathetic division of the ANS. Norepinephrine, as mentioned previously, tends to increase the heart rate of animals. Some studies on mammals have found that norepinephrine not only increases heart rate but also ventricular force and stroke volume. Physiologists conclude that these types of substances may cause muscle fibers to contract more forcefully, regardless of the level of stretch of the myocardium. Such substances are called positive inotropic agents. In today’s laboratory exercise, you will use a force transducer to measure the mechanical activity of the amphibian heart. After determining baseline measurements (heart rate and contractile force), you will subject the heart to various treatments and observe the responses. Specifically, you will:1. Simulate activity of the autonomic nervous system (ANS) by applying Norepinephrine (or Epinephrine) and Acetylcholine to the exposed heart.2. Examine the effect of cold temperature on cardiac muscle activity.3. Identify an unknown pharmacological agent based on it’s mechanism of action (Beta1 or muscarinic agonist or antagonist)Equipment RequiredComputeriWorx data acquisition unit and cable FT-100 force transducer(2) Ring stands and clampsStimulating electrodesSuture threadDissection tray, instruments and pinsSolutions- 50 mL Frog Ringer’s Solution (room temp)- 20 mL Frog Ringer’s Solution (cold / chilled) – do NOT obtain until exercise #3!- 1 mL Norepinephrine (or Epinephrine) equivalent to one full pipette (ask instructor)- 0.25 mL