Name: benefits of cardiac resynchronization therapy in an asynchronous heart failure model induced by left bundle branch ablation and rapid pacing
Uploaded: 2017-12-11T13:00:00
Description: Watch this Scientific Journal Video about Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing at JoVE.com

This work is funded by National Natural Science Foundation of China (81671685) and Shanghai Commission of Health and Family Planning (No. 201440538)

Fifteen male beagle dogs (12 to 18 months old, weighing around 10.0-12.0 kg) were purchased and subjected to experiments. All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996) and were approved by the Animal Care Committee in Zhongshan Hospital, Fudan University. Figure 1 shows the schematic workflow for all protocol steps.
1. Pre-s...

<ol>
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Dilated cardiomyopathy constitutes a major cause of CHF, which is characterized by ventricular dilation, systolic dysfunction with reduced LVEF, and abnormalities of diastolic filling11. Since chronic tachycardia-mediated HF is a recognized clinical condition, rapid pacing of either atrium or ventricle for at least 3 to 4 weeks serves as a frequently used animal model to induce CHF11. Hemodynamic changes occur as soon as 24 h after rapid pacing, with continued deterioration...

Advanced chronic HF is a leading cause of mortality for various cardiovascular diseases. A subset of patients with congestive heart failure (CHF) also develop ventricular conduction discoordination that aggravates symptoms and prognosis. CRT, also referred to as biventricular pacing, has been introduced as an alternative therapy for these patients for over 20 years1,2. Unfortunately, about 20-40% of the patients show poor response to CRT. Since then, many studies have been carried out in order to maximize CRT response3. It is now well recognized that patients with LBBB could benefit more from CRT than those with non-LBBB4, since an LBBB pattern causes a larger magnitude of cardiac dyssynchrony due to asymmetry in the freedom of wall movement between septal and lateral walls. Meanwhile recent studies have begun exploring changes in gene expression and molecular remodeling associated with CRT5. Accompanying the structural reverse remodeling induced by CRT, cellular and molecular reversion to a normal level is of great interest6. Hence, it is essential to establish an optimal model of CHF with isolated LBBB for studying CRT benefits.
Chronic, rapid ventricular pacing was once used to produce CHF in a canine model. RV pacing could undoubtedly produce delayed LV contraction as a model of the LBBB-like contraction pattern. However, this type of functional asynchrony with an intact conduction system may not emulate anatomical LBBB and is not considered an appropriate model for studying CRT performance, the essence of which is to coordinate impaired electrical activation and myocardial contraction. Rapid restoration of LV contractility and partial recovery of LV dimensions after cessation of pacing were also reported7.
Experimental studies have induced chronic LBBB by RF ablation to establish asynchronous ventricular contraction8. A combination of reduction in global pump function and regional invalid mechanical work could exacerbate CHF by generating cardiac inefficiency as well as cardiac remodeling at the tissue, cellular, and molecular levels. In LBBB hearts, workload is lowest in the septum and highest in the LV lateral wall. As a consequence, cardiac remodeling is most pronounced in the lateral wall9. The purpose of the present study is: (i) to advance a stable and chronic HF model with interventricular and intraventricular mechanical asynchrony by means of rapid RV pacing in combination with LBB ablation; (ii) to confirm dyssynchronous HF in our model and CRT benefits in coordinating contraction by two-dimensional speckle tracking echocardiography and aVTI; and (iii) to preliminarily explore cellular reverse remodeling elicited by CRT.

<table>
	<tbody>
		<tr>
			<td>Closed iv catheter system (0.9mm&#215;25mm)</td>
			<td>Becton Dickinson Medical</td>
			<td>5264442</td>
			<td>Used as venous retention needle</td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Sigma-Aldrich Company</td>
			<td>130205</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Pet clipper</td>
			<td>Wuhan Shernbao pet supplies Co., Ltd.</td>
			<td>PGC-660</td>
			<td>For hair shaving</td>
		</tr>
		<tr>
			<td>Electrocardiograph</td>
			<td>Shanghai photoelectric medical electronic instrument Co., Ltd.</td>
			<td>ECG-6511</td>
			<td>For electrocardiogram recording</td>
		</tr>
		<tr>
			<td>Echocardiograph</td>
			<td>GE-Vingmed Ultrasound Company</td>
			<td>VIVID E9</td>
			<td>For echocardiographic assessment</td>
		</tr>
		<tr>
			<td>EchoPAC software</td>
			<td>GE healthcare</td>
			<td>Version201</td>
			<td>Offline analysis</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Shanghai Medical Instrument Co., Ltd</td>
			<td></td>
			<td>Orotracheal intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>SIMS Portex Inc, UK</td>
			<td>274093</td>
			<td>Orotracheal intubation</td>
		</tr>
		<tr>
			<td>Volume cycled respirator</td>
			<td>Newport Corporation</td>
			<td>C100</td>
			<td>Artificial ventilation</td>
		</tr>
		<tr>
			<td>HeartStart XL Defibrillator/Monitor</td>
			<td>Philips Medical Systems</td>
			<td>M4735A</td>
			<td>Electrocardiogram monitor during operation</td>
		</tr>
		<tr>
			<td>Benzalkonium Bromide Tincture</td>
			<td>Shanghai Yunjia Pharmaceutical Co., Ltd.</td>
			<td>H31022694</td>
			<td>Used for skin disinfection</td>
		</tr>
		<tr>
			<td>Rib retractor</td>
			<td>Shanghai Medical Instrument Co., Ltd.</td>
			<td></td>
			<td>For thoracotomy</td>
		</tr>
		<tr>
			<td>4-0 suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>24L1005</td>
			<td>Suture of LV epicardial electrode</td>
		</tr>
		<tr>
			<td>2-0/T suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>11M0505</td>
			<td>Suture of pacing leads, fascia, vessels, etc.</td>
		</tr>
		<tr>
			<td>0-suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>11P0501</td>
			<td>Skin suture</td>
		</tr>
		<tr>
			<td>penicillin powder</td>
			<td>North China Pharmaceutical Co., Ltd.</td>
			<td>F6034105</td>
			<td></td>
		</tr>
		<tr>
			<td>DSA X-ray machine</td>
			<td>Philips</td>
			<td>Allura Xper FD10</td>
			<td>X-ray for fluoroscopy</td>
		</tr>
		<tr>
			<td>LV pacing electrode</td>
			<td>Medtronic, Inc.</td>
			<td>LBT 4965</td>
			<td></td>
		</tr>
		<tr>
			<td>RV pacing electrode</td>
			<td>St. Jude Medical</td>
			<td>Tendril 1888</td>
			<td></td>
		</tr>
		<tr>
			<td>RA pacing electrode</td>
			<td>St. Jude Medical</td>
			<td>IsoFlex 1642T</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacemaker pulse generator</td>
			<td>Medtronic, Inc.</td>
			<td>Enpulse E2DR01</td>
			<td>For rapid RV pacing</td>
		</tr>
		<tr>
			<td>CRT pulse generator</td>
			<td>St. Jude Medical</td>
			<td>Anthem PM 3212</td>
			<td>For CRT performance</td>
		</tr>
		<tr>
			<td>Multi-channel electrophysiologic recorder</td>
			<td>GE Medical Systems</td>
			<td>2003232-004</td>
			<td>For surface and intracardiac electrogram</td>
		</tr>
		<tr>
			<td>Catheter input module</td>
			<td>GE Medical Systems</td>
			<td>301-00202-08</td>
			<td>Multiple pole switches for stimulation or recording</td>
		</tr>
		<tr>
			<td>Radiofrequency generator</td>
			<td>Johnson-Johnson Company</td>
			<td>ST-4460</td>
			<td>For RF current delivery</td>
		</tr>
		<tr>
			<td>Cordless return electrode</td>
			<td>Covidien</td>
			<td>E7509</td>
			<td>For current circuit formation</td>
		</tr>
		<tr>
			<td>Cordis 6-Fr sheath</td>
			<td>Johnson-Johnson Company</td>
			<td>504-606X</td>
			<td>Access for mapping catheter</td>
		</tr>
		<tr>
			<td>Cordis 7-Fr sheath</td>
			<td>Johnson-Johnson Company</td>
			<td>504-607X</td>
			<td>Access for mapping and ablation catheter</td>
		</tr>
		<tr>
			<td>6-Fr quadripolar catheter</td>
			<td>Johnson-Johnson Company</td>
			<td>F6QRA005RT</td>
			<td>Mapping catheter</td>
		</tr>
		<tr>
			<td>7-Fr 4mm-tip steerable ablation catheter</td>
			<td>St. Jude Medical</td>
			<td>402823</td>
			<td>Mapping and ablation catheter</td>
		</tr>
		<tr>
			<td>Prucka Cardio-Lab&#174;2000</td>
			<td>GE Medical Systems</td>
			<td>6.9.00.000</td>
			<td>Software package for electrogram recording</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Haitong Pharmaceutical Co., Ltd</td>
			<td>160505</td>
			<td>Anticoagulant during catheter ablation</td>
		</tr>
		<tr>
			<td>Digital image analysis system</td>
			<td>Leica Microsystems</td>
			<td>Qwin V3</td>
			<td>For histologic analysis</td>
		</tr>
	</tbody>
</table>

benefits of cardiac resynchronization therapy in an asynchronous heart failure model induced by left bundle branch ablation and rapid pacing

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

Successful LBB Ablation:
Figure 2 represents a typical surface and intracardiac electrogram in the course of catheter ablation. The mean LBP-V measured is 18.8 &#177;2.8 ms, which was about 10 ms shorter than the baseline H-V interval (28.8 &#177;2.6 ms, p &#60;0.01). The QRS duration prolonged from 59.2 &#177;6.8 ms to 94.2 &#177;8.6 ms (p &#60;0.01) after LBB ablation. The loss of t...

Watch this Scientific Journal Video about Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing at JoVE.com

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

The overall goal of this experiment is to describe a feasible and valid method to establish a chronic asynchronous heart failure model for studying benefits of cardiac resynchronization therapy. Rapid pacing together with left bundle branch ablation helps to establish a stable heart failure model for studying CRT performance, including echocardiographic parameters, molecular and biologic modifications. The key point of this technique is the left bundle branch ablation, where you prevent the cardiac function recovery on this continuation of rapid pacing.We first had the idea for this method when we found a significant recovery from heart failure once the rapid pacing was terminated. The epicardial left ventricle lead implantation is performed by mister Yao Ruiming, technician from our laboratory who is expert at this procedure. After preparing the canine for the LV pacing electrode implantation, begin with a transverse incision from the left parasternal line at the fourth intercostal space in a right lateral decubitus position.After a blunt dissection of the thoracic muscle, open the left pleural cavity at the fourth intercostal space using sharp dissection. Use a rib retractor to hold open the intercostal space. Next, carefully incise the lateral pericardium and open it using stay sutures to fully expose the LV lateral wall.Then, using a 4-0 suture, secure the LV pacing electrode to the myocardium in one stich. Now, measure the electrode lead parameters. Check that the pacing threshold and other parameters are correct, and then proceed by removing the stay sutures and closing the pericardium with two stitches.Now, use two pericostal sutures to approximate the fourth and fifth ribs using size 0 suture and close the intercostal fascia muscularis with five stitches of 2-0/T sutures. Be sure to inflate the lungs adequately before the last suture. Then, put the muscle layers back in place.Next, build a subcutaneous tunnel above the deep fascia from precordial area to the left cervix. Then, pull the terminal pin at the lead through the tunnel and suture the lead around an insulation sleeve to the fascia. Embed the lead locally.To complete the surgery, provide penicillin and close the two incisions. Implant the RA and RV pacing electrodes two weeks after the LV electrode implantation, by which time the animal should have recovered from the thoracotomy. Carry out the operation in a cardiac catheterization surgery room equipped with the fluoroscopy apparatus.After preparing the animal for surgery, begin with a small vertical incision close to the preceding wound on the left side of the cervical area. Then, using blunt dissection, separate the fascia to expose the left external jugular vein. Be sure to carefully separate the vein from the connective tissues.With the aid of stay sutures, cut a small hole on the vein using iris scissors. Then, using a vein pick, insert a passive J-shaped RA lead and active RV lead into the left external jugular vein. Now, under fluoroscopy, advance the RV lead to the lower right atrium or inferior vena cava.Then withdraw the straight stylet from the RV lead and insert a J-shaped stylet. With the help of the J-shaped stylet, introduce the lead across the tricuspid valve and into the outflow tract. Then, slowly withdraw both the lead and the stylet, allowing the lead tip to prolapse toward the RV apex.Now, replace the curved stylet with a straight one and advance the lead towards the apex. Then, slowly withdraw the stylet a little while keeping the RA lead directed toward the high anterior atrium. Slowly withdraw the straight stylet.Then, retract the tip of the J-shaped lead into the appendage. A characteristic to-and-fro motion of the electrode with atrial activity may be observed. After obtaining satisfactory lead parameters and checking the stability of both leads, tighten the suture proximal to the venotomy.Then, tie down both leads to the underlying deep fascia around the suture sleeves. Then, check the positioning of the leads under fluoroscopy before proceeding with the pulse generator implantation. For sham surgeries, skip this procedure.To implant the pulse generator, first dissect bluntly with the aid of a curved clamp to make a pocket for the pulse generator near the venous entry just above the fascia layer and below the subcutaneous fat. Next, clean and dry the lead pins. Cover the terminal pin at the atrial lead with an insulating sleeve, then suture the lead to the floor of the pocket.Then, insert the ventricular lead into a pacemaker pulse generator and tighten it with a distal connector pin past the set screws of the generator. Now, place the generator into the pocket and quell the redundant bleeds beneath the device. Then, secure the generator to the fascia using another suture to the tie-down hole.Now, perform a fluoroscopic examination of the entire system and check for homeostasis. To complete the surgery, sprinkle 200, 000 units of penicillin into the pocket and suture the superficial fascia layer closed using 2-0/T sutures. Then, approximate the skin edges with both sutures.Carry out the catheter ablation under the guidance of fluoroscopy. Have a multi-general electrophysiological recorder prepared for simultaneous surface and intracardiac electrogram recording. Shave the hair off the chest, back and right inguinal region.Keep the animal in a supine position. The first surgical steps are to insert sheets into the femoral vein and femoral artery for catheter placement. This is described in detail in the text protocol.To proceed, advance a six French steerable quadripolar catheter into the femoral vein and connect the end of the catheter to the recorder. Then, pass the catheter into the right atrium and across the tricuspid valve until it is clearly in the right ventricle. Using a right anterior oblique 30-degree view, withdraw the catheter across the tricuspid orifice until the atrial potential appears and it increases in amplitude.When the atrial and ventricular potentials are approximately equal in size, a biphasic or triphasic deflection will appear between them. This represents the right side of His bundle potential. Now, introduce a number seven French formula meter tip steerable ablation catheter into the femoral artery to map the left bundle branch potential.Then, connect the ablation catheter to the RF generator and the multichannel electrophysiological recorder. Using a right anterior oblique 30-degree view, pass the arterial catheter retrogradely across the aortic valve and advance it into the left ventricle. There, deflect the catheter tip toward the intraventricular septum where it should remain in contact with the septum.Now, using a left anterior oblique 45-degree view, slowly withdraw the catheter along the septum until the left side of His bundle potential is recorded between the atrial and ventricular electrogram just below the aortic valve. Then, slightly advance the catheter along the septum and manipulate the tip to identify a discrete left bundle potential, which is recorded beneath the aortic valve. It is usually located one to one and a half centimeters inferior to left side of His bundle recording site.When the potential to ventricular electrogram interval is 10 milliseconds or so shorter than the HV interval and the AV electrogram ratio of less than one to 10, then the left bundle potential has been identified. Next, perform the ablation with the RF generator unless it is a sham surgery. Deliver 500 kilohertz of unmodulated sine wave energy at 30 to 40 watts to achieve a target temperature of 60 degrees Celsius at the electrode-tissue interface.When a typical LBBB QRS morphology appears on the surface electrogram, continue the energy application. Then, observe the surface electrogram for a stabilization period of 30 minutes. To complete the procedure, remove both catheters.Then, remove the sheets and tighten the suture knots rapidly to prevent hemorrhaging. Lastly, provide penicillin and close the incisions. The typical surface and intracardiac electrogram in the course of catheter ablation shows the mean LVPV to be about ten milliseconds shorter than the baseline HV interval, and after the LBB ablation, the QRS duration is prolonged.Baseline echocardiographic parameters showed no difference between the groups. After the experimental intervention, the heart failure group had an obvious deterioration in cardiac function from which no measured parameter improved over eight weeks of observation. However, with eight weeks of CRT, the left ventricle and diastolic volume returned to normal, as did the left ventricle and systolic volume.CRT also returned the lost left ventricle ejection fraction. Longitudinal strain curves of six segments from each plain made by speckle tracking analysis showed a measurable increase in the dispersion of the time to peak longitudinal strain in the heart failure group. By contrast, the therapy corrected the asynchrony.This was observable in the four-chamber view, the two-chamber view and in the long axis view. Furthermore, the heart failure animals presented a significantly lower aortic velocity time integral than the shams. This metric was significantly increased by the CRT.Histology revealed a remarkable decrease in cardiomyocyte diameter and a significant increase in the collagen volume fraction in the heart failure group. However, eight weeks of CRT provided a significant restoration of these metrics, indicating that the therapy invoked remodeling of the tissue. After watching this video, you should have got a good understanding of how to establish a chronic heart failure with isolated LBBB for studying CRT benefits.Following the left bundle branch ablation, apart from rapid pacing, we can still use some other method to induce heart failure such as a coronary ligation to induce an ischemic heart failure. This well established technique was paved the way for researches to study cardiovascular diseases in large animal models of heart failure.

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Research

JoVE Journal

Biology

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Real-Time Measurements of Calcium and Contractility Parameters in hiPSC-CMs

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

Author Spotlight: Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Exploring HR-Dependent Cardiac Performance Using an Atrial-Pacing Protocol

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.

Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function&#160;in mouse models of heart failure.

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While ...

Instrument and Animal Preparation for Catheterization in a Murine Heart Failure Model

To begin, place a conductance catheter in normal saline, then connect it to an MPVS module, comprising the PowerLab 8/35 and a pressure volume unit. Electronically calibrate the pressure and volume by recording the predetermined pressure and volume parameters on the MPVS module. For the murine heart failure model, anesthetize the animal, and ensure that the surgical site is adequately shaved and disinfected.Expose the trachea and insert an endotracheal tube into it. Locate the internal jugular vein beneath the sternocleidomastoid muscle. Then insert a central venous catheter consisting of PE10 Silastic connected to a 30 gauge needle into the jugular vein.Administer a bolus infusion of five to six microliters per gram of body weight of 10%Albumin in sodium chloride for about three minutes. Follow this with a constant infusion rate of five to 10 microliters per minute.