This work was funded by the National Institutes of Health, grant numbers R01NS100954 and R01NS099188.

The authors have nothing to disclose.

Practicing and mastering the SAN dissection process is imperative since the tissue is fragile and healthy tissue is necessary for a successful recording. During the SAN dissection, correct orientation is essential to obtain the proper region of tissue. However, the original orientation of the heart can be easily lost during the dissection process, which complicates this endeavor. Therefore, to ensure the proper left-right orientation, the atria should be visually inspected. Typically, the right atrium tends to be more tr...

The heart is a complex organ governed by both cardiac-intrinsic and extrinsic influences such as those that originate in the brain. The sinoatrial node (SAN) is a defined region in the heart that houses the pacemaker cells (also referred to as sinoatrial cells, or SA cells) responsible for the initiation and perpetuation of the mammalian heartbeat1,2. The intrinsic heart rate is the rate driven by the pacemaker cells without influence by other cardiac or neuro-humoral influences, but traditional measures of heart rate in humans and live animals, such as electrocardiograms, reflect both the pacemaker and neural influences on the heart. The most notable neural influence on SA cells is from the autonomic nervous system, which constantly modulates firing patterns to meet the physiological requirements of the body3. Supporting this idea, both sympathetic and parasympathetic projections can be found near the SAN4. The intrinsic cardiac nervous system (ICNS) is another important neural influence where ganglionated plexi, specifically in the right atria, innervate and regulate the activity of the SAN5,6.
Understanding pacemaking deficits is clinically important, as dysfunction can underlie many cardiac disorders, as well as contribute to the risk of other complications. Sick sinus syndrome (SSS) is a category of diseases characterized by dysfunction of the sinoatrial node which impedes proper pacemaking7,8. SSS can present with sinus bradycardia, sinus pauses, sinus arrest, sinoatrial exit block, and alternating bradyarrythmias and tachyarrhythmias9 and can lead to complications including increased risk of embolic stroke and sudden death8,10. Those with Brugada syndrome, a cardiac disorder marked by ventricular fibrillation with an increased risk of sudden cardiac death, are at greater risk for arrhythmogenic events if they also have comorbid SAN dysfunction11,12. Sinoatrial dysfunction may also have physiological consequences beyond the heart. For example, SSS has been observed to trigger seizures in a patient due to cerebral hypoperfusion13.
To identify sinoatrial pacemaking deficits, intrinsic heart rates need to be determined by measuring the activity of the SAN without the influence of the autonomic nervous system or humoral factors. Clinically, this can be approximated by pharmacological autonomic blockade14, but this same technique can also be applied in mammalian models to study intrinsic cardiac function15,16. While this approach blocks a large portion of contributing neural influences and allows for in vivo cardiac examination, it does not completely eliminate all extrinsic influences on the heart. Another research technique used to study intrinsic cardiac function in animal models is isolated heart recordings using Langendorff-perfused hearts, which typically involve measurements using electrograms, pacing, or epicardial multielectrode arrays17,18,19,20. While this technique is more specific to cardiac function since it involves removing the heart from the body, the measurements may still be influenced by mechano-electric autoregulatory mechanisms that could influence intrinsic heart rate measurements21. The isolated heart recordings may also still be influenced by autonomic regulation through the ICNS5,6,22,23. Furthermore, maintaining a physiologically relevant temperature of the heart, which is critical for cardiac function measurements, can be difficult in isolated heart approaches20. A more direct method to study SAN function is to specifically isolate SAN tissue and measure its activity. This can be accomplished through SAN strips (isolated SAN tissue) or isolated SAN pacemaker cells24,25. Both require a high degree of technical training, as the SAN is a very small and highly defined region, and cell isolation poses an even greater challenge as dissociation can damage the overall health of the cell if not performed correctly. Furthermore, these techniques require expert electrophysiological skills in order to successfully record from the tissue or cells using individual recording microelectrodes.
In this protocol, we describe a technique to record the SAN in vitro by using a microelectrode array (MEA) to obtain intrinsic heart rate measurements. This approach has the advantage of making highly specific electrophysiological recordings accessible to researchers lacking intensive electrophysiological skillsets. MEAs have previously been used to study cardiomyocyte function in primary cardiomyocyte cultures26,27,28,29,30,31,32, cardiac sheets33,34,35,36,37,38,39, and tissue whole mounts40,41,42,43,44,45,46,47. Previous work has also been done to examine field potentials in SAN tissue41,42. Here, we provide a methodology to use the MEA to record and analyze murine intrinsic SAN firing rates. We also describe how this technique can be used to test pharmacological effects of drugs on SAN intrinsic firing rates by providing a sample experiment showing the effects of 4-aminopyridine (4-AP), a voltage-gated K+ channel blocker. Using defined anatomical landmarks, we can accurately record the SAN without having to perform the extensive tissue dissections or cell isolations required in other methods. While the MEA can be cost-prohibitive, the recordings provide highly specific and reliable measures of pacemaking that can be used in a vast array of clinical and physiological research applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>4-Aminopyridine</td>
			<td>Sigma</td>
			<td>A78403-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>22 gauge syringe needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5A</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>23 gauge syringe needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-6C</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>60mm Petri Dishes</td>
			<td>Genesee Scientific</td>
			<td>32-105G</td>
			<td></td>
		</tr>
		<tr>
			<td>500mL Pyrex Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1C</td>
			<td>Used to store solutions</td>
		</tr>
		<tr>
			<td>1000 mL Pyrex Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td>Used to store solutions</td>
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		<tr>
			<td>Bone Forceps</td>
			<td>Fine Science Tools</td>
			<td>16060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen (95% O2, 5% CO2)</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Castroviejo Scissors, 4&#34;</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
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		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition PC</td>
			<td></td>
			<td></td>
			<td>CPU: Intel Xeon or Intel Core i7, Memory: 8GB, HDD: 1TB, Graphic Card: NVIDIA or On-board, Screen: 1920x1080</td>
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		<tr>
			<td>Dissection Microscope</td>
			<td>Jenco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #2 Laminectomy Forceps</td>
			<td>Fine Science Tools</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Chamber</td>
			<td>Grainger</td>
			<td>49WF30</td>
			<td>Used for mouse euthanization</td>
		</tr>
		<tr>
			<td>Harp Anchor Kit</td>
			<td>Warner Instruments</td>
			<td>&#160;SHD-22CL/15 WI 64-0247</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Chemicals</td>
			<td>SA48-4</td>
			<td>Used for pH balancing</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Aurobindo Pharma Limited IDA, Pashamylaram</td>
			<td>NDC 63739-953-25</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Motic</td>
			<td>AE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tape</td>
			<td>Fisher Scientific</td>
			<td>15-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Light for Dissection Microscope</td>
			<td>Dolan-Jenner</td>
			<td>MI150DG 660000391014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>208337-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Head Amplifier</td>
			<td>MED64</td>
			<td>MED-A64HE1S</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Main Amplifier</td>
			<td>MED64</td>
			<td>MED-A64MD1A</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Perfusion Cap</td>
			<td>MED64</td>
			<td>MED-KCAP01</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Perfusion Pipe Holder Kit</td>
			<td>MED64</td>
			<td>MED-KPK02</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 ThermoConnector</td>
			<td>MED64</td>
			<td>MED-CP04</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>&#160;Warner Instruments</td>
			<td>640246</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode array (MEA)</td>
			<td>Alpha Med Scientific</td>
			<td>MED-R515A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobius Software</td>
			<td>WitWerx Inc.</td>
			<td></td>
			<td>Specific software for the MED64</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Chemicals</td>
			<td>S320-500</td>
			<td>Used for pH balancing</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Ultigiene</td>
			<td>NDC 50989-885-17</td>
			<td></td>
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		<tr>
			<td>Paint Brush</td>
			<td>Fisher Scientific</td>
			<td>NC1751733</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Genesee Scientific</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Gilson</td>
			<td>F155009</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171-298</td>
			<td>1/8&#39;&#39; Interior Diameter</td>
		</tr>
		<tr>
			<td>Polyethyleneimine</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgruard Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S6566</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate</td>
			<td>Sigma</td>
			<td>S9640</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14074-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipets (3mL graduated)</td>
			<td>Samco Scientific</td>
			<td>225</td>
			<td></td>
		</tr>
	</tbody>
</table>

microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

After allowing the tissue to acclimate in the dish for 15 min, 10 one-min traces are recorded. Our current protocol records activity for over an hour, but we have recorded stable firing patterns for &#8805;4 h in unpublished data not shown here. If an experimental preparation is good for data collection, each recording channel should exhibit consistent and evenly spaced recurring waveforms (i.e., spikes) of uniform shape for a given channel (Figure 11D). These waveforms correspond to individ...

Watch this Scientific Journal Video about Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice at JoVE.com

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

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2.9K Views.  Southern Methodist University. This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

Video: Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology

Participation in organized sports makes a significant contribution to youth development, but places youth at a higher risk for sustaining a concussion. To date, return-to-activity decision-making has been anchored in the monitoring of self-reported concussion symptoms and neurocognitive testing. However, multi-modal assessments that corroborate objective physiological measures with traditional subjective symptom reporting are needed and can be valuable. Heart rate variability (HRV) is a non-invasive physiological indicator of the autonomic nervous system, capturing the reciprocal interplay between the sympathetic and parasympathetic nervous systems. There is a dearth of literature exploring the effect of concussion on HRV in youth athletes, and developmental differences preclude the application of adult findings to a pediatric population. Further, the current state of HRV methodology has primarily included short-term (5-15 min) recordings, by using resting state or short-term physical exertion testing to elucidate changes following concussion. The novelty in utilizing a 24 h&#160;recording methodology is that it has the potential to capture natural variation in autonomic function, directly related to the activities a youth athlete performs on a regular basis. Within a prospective, longitudinal research setting, this novel approach to quantifying autonomic function can provide important information regarding the recovery trajectory, alongside traditional self-report symptom measures. Our objectives regarding a 24 h recording methodology were to (1) evaluate the physiological effects of a concussion in youth athletes, and (2) describe the trajectory of physiological change, while considering the resolution of self-reported post-concussion symptoms. To achieve these objectives, non-invasive sensor technology was implemented. The raw beat-to-beat time intervals captured can be transformed to derive time domain and frequency domain measures, which reflect an individual&#39;s ability to adapt and be flexible to their ever-changing environment. By using non-invasive heart rate technology, autonomic function can be quantified outside of a traditional controlled research setting.

We demonstrate a 24 h heart rate recording methodology to evaluate the influence of concussion across the recovery trajectory in youth athletes, within an ecologically valid context.

Participation in organized sports makes a significant contribution to youth development, but places youth at a higher risk for sustaining a concussion. To ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...