This work was supported by Onderzoekstoelagen grant OT/13/090 of the KU Leuven and by grant G0A3114N of the FWO-Vlaanderen.

NOTE: All experimental procedures described in this section were approved by the Institutional Animal Care and Research Advisory Committee of the Katholieke Universiteit Leuven (Project: 154/2013-B De Geest).
1. Permanent Ligation of the Left Anterior Descending Coronary Artery
<ol>
	<li>Anesthetize the mouse by intraperitoneal administration of 40&#160;mg/kg to 70 mg/kg of sodium pentobarbital. Ensure the mouse reaches its proper plane of anesthesia when it no longer reacts a firm toe pinch. Always confirm proper anesthetization this way prior to any surgical procedure or intervention. Use lubricating ophthalmic ointment to prevent dryness of the cornea while under anesthesia.&#160;Provide preoperative analgesia 2-4 hr before the start of the procedure (buprenorphine 0.05 mg/kg SQ).
	<ol>
		<li>Apply consistent aseptic techniques during survival surgery. Implement procedures that inhibit to a maximally possible extent microbial contamination so that significant infection or suppuration does not occur. These procedures include use of sterile instruments and sterile materials, disinfection of the surgical area, and removal of fur/hair over the surgical site and disinfection of this site.</li>
	</ol>
	</li>
	<li>Intubate the mouse with a self-prepared blunted 20-gauge needle.
	<ol>
		<li>Put the mouse in supine position with hyperextension of the head.
		<ol>
			<li>Focus the light on the neck region. Lift the tongue with a blunted pincet. The entrance of the larynx can be clearly seen.</li>
			<li>Pass the blunted needle through the larynx into the trachea under direct vision. Assess correct intubation by connecting the mouse to the ventilator (stroke volume in &#181;l: 3 x body weight (g) + 155; frequency: 120 strokes per min).</li>
		</ol>
		</li>
		<li>Alternatively, enhance visualization of endotracheal intubation by first carefully exposing the trachea.
		<ol>
			<li>Make a 5 mm mid-neck incision and retract muscle tissue just above the trachea.</li>
			<li>Perform the intubation using a surgical stereomicroscope for direct visualization of the trachea. Lift the tongue and enter the self-prepared blunted 20 gauge needle into the trachea. Confirm correct intubation by connecting the mouse to the ventilator (stroke volume in &#181;l: 3 x body weight (g) + 155; frequency: 120 strokes per min).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Keep the mouse in supine position and fix the mouse with tape. Perform surgery on a heating pad to prevent hypothermia.
	<ol>
		<li>Shave and disinfect the skin with Betadine. Take care that the left hind limb crosses the right hind limb in order to obtain a better view on the left ventricle during surgery.</li>
	</ol>
	</li>
	<li>Make a small transverse cutaneous incision up to the sternum and separate the underlying skin and muscles.</li>
	<li>Pull aside the m. pectoralis minor and m. pectoralis major with a 5-0 silk suture.</li>
	<li>Make an incision in the third intercostal space by inserting a blunted pincet.</li>
	<li>Move the pincet under the intercostal muscles from lateral to medial till the sternum is reached. Puncture the thoracic wall by pushing the pincet from inside to the skin. Complete the thoracotomy by carefully cutting the intercostal muscle just above the pincet with a small scissor. Use this technique to prevent puncturing of the lungs.</li>
	<li>Put a sponge immersed in 0.9% NaCl into the cavity to protect the lungs. Introduce a wound spreader (chest retractor) into the intercostal space to obtain exposure of the left side of the heart. As of now, the left atrium, the left ventricle, and the left anterior descending coronary artery are visible under the stereomicroscope.</li>
	<li>Perform a ligation of the left anterior descending coronary artery with a single 6-0 prolene ligature about 1 mm under the tip of the left atrium. This is distal from the first diagonal branch. 
	NOTE: Alternatively, 7-0 (0.05 mm diameter) or 8-0 threads (0.04 mm diameter) may be used. The needle is a C-1 13 mm 3/8 circle Taper Point needle. Successful ligation of the left anterior descending coronary artery induces immediate discoloration, resulting in a pale appearing myocardium in the affected territory.</li>
	<li>Remove the wound spreader (chest retractor).
	<ol>
		<li>Place three 6-0 Ti-Cron sutures around the intercostal space. Before tightening the sutures, remove the sponge from the chest cavity and re-expand the lungs by blocking the outflow of the ventilator. By doing so, the lungs reconnect with the parietal pleura.</li>
		<li>Subsequently, pull the sutures tight and repeat re-expansion by pressing down on the chest. Confirm the successful closure of the thorax using a small amount of saline (no air bubbles must be seen when applying pressure on the chest).</li>
		<li>Look through the intercostal muscle to confirm normal expansion of the lungs. Reposition both pectoralis muscles, serving as an extra barrier for prevention of a pneumothorax.</li>
	</ol>
	</li>
	<li>Close the skin with 5-0 silk-sutures.</li>
	<li>Disconnect the mouse from the ventilator and allow recovery on the heating pad. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Consistently provide postoperative analgesia (buprenorphine 0.05 mg/kg SQ BID for at least 48 hr after surgery).&#160;</li>
</ol>
2. In vivo Invasive Hemodynamic Measurements in Mice
<ol>
	<li>Before the procedure, submerge the 1.0 French Millar pressure catheter in sterile water at 37 &#176;C for at least 30 min to minimize signal drift. Electronically calibrate the pressure sensor at 0 mm Hg and 100 mm Hg and record data at 2,000 Hz.</li>
	<li>Perform anesthesia by intraperitoneal administration of 1.4 g/kg urethane. Check that the mouse reaches its proper plane of anesthesia when it no longer reacts a firm toe pinch.</li>
	<li>Place the anesthetized mouse in supine position. Secure its limbs with tape. Maintain body temperature with a heating pad and monitor with a rectal probe. Shave the neck region and make a midline incision in the neck region to expose the thyroid gland.</li>
	<li>Fix the neck with bent needles.</li>
	<li>Pull aside the salivary gland and expose the right common carotid artery. The vagus nerve, which resembles a white thread, lies along the artery. Carefully separate the carotid artery from the vagus nerve using a curved forceps.</li>
	<li>Pass a curved forceps under the right common carotid artery to separate it from other tissues. Remove connective tissue around the artery.</li>
	<li>Pass two 6-0 silk wires under the right common carotid artery. Make a tight knot on the top wire, which is placed towards the head, close, and fix with a kocher (distal occlusive ligation). Pass the proximal wire twice from left to right and fix with 2 kochers (proximal non-occlusive wire).</li>
	<li>Keep the carotid artery moist by dropping sterile 0.9% NaCl. Dry off excess liquid with cotton buds.</li>
	<li>Make an incision in the right common carotid artery with a 26-gauge needle between the distal ligation and the proximal non-occlusive wire.</li>
	<li>Introduce the pressure sensor into the artery. Verify that there is no blood loss. Gently push the 1.0 French Millar pressure catheter forward and adjust the proximal non-occlusive wire in such a way that the catheter can pass carefully through the wire under the clavicle.
	<ol>
		<li>Minimize blood loss during adjustment of the proximal non-occlusive wire. Do not compress the pressure sensor too much while advancing since it is very fragile. Since the proximal wire must not occlude the artery, the vessel should remain filled with blood.</li>
	</ol>
	</li>
	<li>Start recording the pressure signal. An arterial pressure signal fluctuates in a healthy mouse between a diastolic pressure of 60 - 70 mm Hg and a systolic pressure of 100 - 120 mm Hg.</li>
	<li>Direct the catheter via the innominate artery and via the aorta into the left ventricle. The ventricular pressure fluctuates between 0 mm Hg and 100 - 120 mm Hg. Allow the catheter to stabilise inside the left ventricle. Record the signal for 30 min to 60 min depending on the experimental requirements.</li>
	<li>After completion of the experiment, soak the catheter in Alconox 1% for 30 min. Wash the catheter with Milli-Q water. Store the catheter in a foam block.</li>
	<li>Retrieve data from the recording software for further analysis.
	<ol>
		<li>For data analysis, consider a time interval where the pressure signal is stable. Choose at least 10 consecutive cardiac cycles of the recorded data of interest.</li>
		<li>Use LabChart software version 8.0 or similar to analyse the heart rate, the maximal systolic left ventricular pressure, the minimal diastolic left ventricular pressure, the peak rate of isovolumetric left ventricular contraction (dP/dtmax), the peak rate of isovolumetric left ventricular relaxation (dP/dtmin), the end-diastolic left ventricular pressure, and the time constant of the isovolumetric left ventricular pressure fall (tau)7. 
		NOTE: The end-diastolic pressure corresponds to the pressure at the time-point immediately before the pressure surge induced by isovolumetric contraction. The calculation of tau is based on fitting the left ventricular pressure into a monoexponential decay curve, expressed as P(t)=P0e-t/tau + b, In this formula, P(t) is the left ventricular pressure at time t after the maximal negative value of dP/dt has been reached. The parameter b corresponds to the theoretical asymptote, which in a simplified approach can be assumed to be zero. Enhanced isovolumetric relaxation results in a smaller value of tau.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>He, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+congestive+heart+failure+in+US+men+and+women:+NHANES+I+epidemiologic+follow-up+study.">Risk factors for congestive heart failure in US men and women: NHANES I epidemiologic follow-up study.</a> Arch Intern Med. 161, 996-1002 (2001).</li><li>Anversa, P., Sonnenblick, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+cardiomyopathy:+pathophysiologic+mechanisms.">Ischemic cardiomyopathy: pathophysiologic mechanisms.</a> Prog Cardiovasc Dis. 33, 49-70 (1990).</li><li>Erlebacher, J. A., Weiss, J. L., Weisfeldt, M. L., Bulkley, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+dilation+of+the+infarcted+segment+in+acute+transmural+myocardial+infarction:+role+of+infarct+expansion+in+acute+left+ventricular+enlargement.">Early dilation of the infarcted segment in acute transmural myocardial infarction: role of infarct expansion in acute left ventricular enlargement.</a> J Am Coll Cardiol. 4, 201-208 (1984).</li><li>Shimizu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+cardiac+insulin+signaling+exacerbates+systolic+dysfunction+induced+by+pressure+overload+in+rodents.">Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents.</a> J Clin Invest. 120, 1506-1514 (2010).</li><li>Tirziu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+hypertrophy+in+the+absence+of+external+stimuli+is+induced+by+angiogenesis+in+mice.">Myocardial hypertrophy in the absence of external stimuli is induced by angiogenesis in mice.</a> J Clin Invest. 117, 3188-3197 (2007).</li><li>Theilmeier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+lipoproteins+and+their+constituent,+sphingosine-1-phosphate,+directly+protect+the+heart+against+ischemia/reperfusion+injury+in+vivo+via+the+S1P3+lysophospholipid+receptor.">High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor.</a> Circulation. 114, 1403-1409 (2006).</li><li>Weiss, J. L., Frederiksen, J. W., Weisfeldt, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+determinants+of+the+time-course+of+fall+in+canine+left+ventricular+pressure.">Hemodynamic determinants of the time-course of fall in canine left ventricular pressure.</a> J Clin Invest. 58, 751-760 (1976).</li><li>Bohl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+approach+for+quantification+of+in+vivo+ischemia-reperfusion+injury+in+the+mouse+heart.">Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart.</a> Am J Physiol Heart Circ Physiol. 297, 2054-2058 (2009).</li><li>Van Craeyveld, E., Jacobs, F., Gordts, S. C., De Geest, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+gene+transfer+in+hypercholesterolemic+mice+improves+cardiac+function+after+myocardial+infarction.">Low-density lipoprotein receptor gene transfer in hypercholesterolemic mice improves cardiac function after myocardial infarction.</a> Gene Ther. 19, 860-871 (2012).</li><li>Gordts, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+selective+HDL-raising+gene+transfer+on+survival,+cardiac+remodelling+and+cardiac+function+after+myocardial+infarction+in+mice.">Beneficial effects of selective HDL-raising gene transfer on survival, cardiac remodelling and cardiac function after myocardial infarction in mice.</a> Gene Ther. 20, 1053-1061 (2013).</li><li>Junqueira, L. C., Bignolas, G., Brentani, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picrosirius+staining+plus+polarization+microscopy,+a+specific+method+for+collagen+detection+in+tissue+sections.">Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.</a> Histochem J. 11, 447-455 (1979).</li><li>Patten, R. D., Hall-Porter, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+heart+failure:+development+of+novel+therapies,+past+and+present.">Small animal models of heart failure: development of novel therapies, past and present.</a> Circ Heart Fail. 2, 138-144 (2009).</li><li>Zolotareva, A. G., Kogan, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+experimental+occlusive+myocardial+infarction+in+mice.">Production of experimental occlusive myocardial infarction in mice.</a> Cor Vasa. 20, 308-314 (1978).</li><li>Michael, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+ischemia+and+reperfusion:+a+murine+model.">Myocardial ischemia and reperfusion: a murine model.</a> Am J Physiol. 269, 2147-2154 (1995).</li><li>Salto-Tellez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+in+the+C57BL/6J+mouse:+a+quantifiable+and+highly+reproducible+experimental+model.">Myocardial infarction in the C57BL/6J mouse: a quantifiable and highly reproducible experimental model.</a> Cardiovasc Pathol. 13, 91-97 (2004).</li><li>Fernandez, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+arteries+of+the+C57BL/6+mouse+strains:+implications+for+comparison+with+mutant+models.">The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.</a> J Anat. 212, 12-18 (2008).</li><li>Kumar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mouse+coronary+anatomy+and+myocardial+infarction+consequent+to+ligation.">Distinct mouse coronary anatomy and myocardial infarction consequent to ligation.</a> Coron Artery Dis. 16, 41-44 (2005).</li><li>Clauss, S. B., Walker, D. L., Kirby, M. L., Schimel, D., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+coronary+arteries+in+wildtype+and+connexin43+knockout+mice.">Patterning of coronary arteries in wildtype and connexin43 knockout mice.</a> Dev Dyn. 235, 2786-2794 (2006).</li><li>Icardo, J. M., Colvee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+course+of+the+coronary+arteries+in+normal+mice+and+in+iv/iv+mice.">Origin and course of the coronary arteries in normal mice and in iv/iv mice.</a> J Anat. 199, 473-482 (2001).</li><li>Yoldas, A., Ozmen, E., Ozdemir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+description+of+the+coronary+arteries+in+Swiss+albino+mice+(Mus+musculus).">Macroscopic description of the coronary arteries in Swiss albino mice (Mus musculus).</a> J S Afr Vet Assoc. 81, 247-252 (2010).</li><li>James, T. N., Burch, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+supply+of+the+human+interventricular+septum.">Blood supply of the human interventricular septum.</a> Circulation. 17, 391-396 (1958).</li><li>Gao, X. M., Xu, Q., Kiriazis, H., Dart, A. M., Du, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+post-infarct+ventricular+rupture:+time+course,+strain-+and+gender-dependency,+tensile+strength,+and+histopathology.">Mouse model of post-infarct ventricular rupture: time course, strain- and gender-dependency, tensile strength, and histopathology.</a> Cardiovasc Res. 65, 469-477 (2005).</li><li>Muthuramu, I., Jacobs, F., Singh, N., Gordts, S. C., De Geest, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+homocysteine+lowering+gene+transfer+improves+infarct+healing,+attenuates+remodelling,+and+enhances+diastolic+function+after+myocardial+infarction+in+mice.">Selective homocysteine lowering gene transfer improves infarct healing, attenuates remodelling, and enhances diastolic function after myocardial infarction in mice.</a> PLoS One. 8, 63710 (2013).</li><li>Eaton, L. W., Weiss, J. L., Bulkley, B. H., Garrison, J. B., Weisfeldt, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cardiac+dilatation+after+acute+myocardial+infarction:+recognition+by+two-dimensional+echocardiography.">Regional cardiac dilatation after acute myocardial infarction: recognition by two-dimensional echocardiography.</a> N Engl J Med. 300, 57-62 (1979).</li><li>Erlebacher, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+effects+of+acute+infarct+dilation+on+heart+size:+a+two+dimensional+echocardiographic+study.">Late effects of acute infarct dilation on heart size: a two dimensional echocardiographic study.</a> Am J Cardiol. 49, 1120-1126 (1982).</li><li>Schuster, E. H., Bulkley, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+transmural+myocardial+infarction:+a+pathophysiologic+factor+in+cardiac+rupture.">Expansion of transmural myocardial infarction: a pathophysiologic factor in cardiac rupture.</a> Circulation. 60, 1532-1538 (1979).</li><li>Jugdutt, B. I., Michorowski, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+infarct+expansion+in+rupture+of+the+ventricular+septum+after+acute+myocardial+infarction:+a+two-dimensional+echocardiographic+study.">Role of infarct expansion in rupture of the ventricular septum after acute myocardial infarction: a two-dimensional echocardiographic study.</a> Clin Cardiol. 10, 641-652 (1987).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nat Protoc. 3, 1422-1434 (2008).</li><li>Vanden Bergh, A., Flameng, W., Herijgers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+ventricular+contractility+in+mice:+influence+of+load+and+sensitivity+to+changes+in+inotropic+state.">Parameters of ventricular contractility in mice: influence of load and sensitivity to changes in inotropic state.</a> Pflugers Arch. 455, 987-994 (2008).</li></ol>

None of the authors reports competing financial interests.

Chronic changes in myocardial structure and function, the development of left ventricular dysfunction, and progression to heart failure can be investigated in several murine models12. Cardiac remodelling and dysfunction can be induced by myocardial injury or by pressure overload secondary to transverse aortic constriction, or may be investigated in genetic models of dilated cardiomyopathy12. Obviously, the most pronounced benefit of murine models is the availability of a great number of transgenic a...

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with the cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure1.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. In other words, ventricular remodelling concerns an alteration of the left ventricular architecture. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload (e.g., mitral insufficiency). Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure.
Loss of myocardial tissue following acute myocardial infarction results in a decreased systolic ejection and an increased left ventricular end-diastolic volume and pressure. The Frank-Starling mechanism, implying that an increased end-diastolic volume results in an increased pressure developed during systole, may help to restore cardiac output. However, the concomitant increased wall stress may induce regional hypertrophy in the non-infarcted segment, whereas in the infarcted area expansion and thinning may occur. Experimental animal studies show that the infarcted ventricle hypertrophies and that the degree of hypertrophy is dependent on the infarct size2. 
The loss of myocardial tissue following acute myocardial infarction results in a sudden increase in loading conditions. Post-infarct remodelling occurs in the setting of volume overload, since the stretched and dilated infarcted tissue increases the left ventricular volume. An increased ventricular volume not only implies increased preload (passive ventricular wall stress at the end of diastole) but also increased afterload (total myocardial wall stress during systolic ejection). Afterload is increased since the systolic radius is increased. Therefore, ventricular remodelling post-myocardial infarction is characterized by mixed features of volume overload and pressure overload.
The myocardium consists of 3 integrated components: cardiomyocytes, extracellular matrix, and the capillary microcirculation. All 3&#160;components are involved in the remodelling process. Matrix metalloproteinases produced by inflammatory cells induce degradation of intermyocyte collagen struts and cardiomyocyte slippage. This leads to infarct expansion characterized by the disproportionate thinning and dilatation of the infarct segment3. In later stages of remodelling, interstitial fibrosis is induced, which negatively affects the diastolic properties of the heart. 
The vascular and cardiomyocyte compartment in the myocardium should remain balanced in the process of ventricular remodelling to avoid tissue hypoxia4,5. Whether hypertrophy progresses to heart failure or not may be critically dependent on this balance between the vascular and cardiomyocyte compartment in the myocardium. 
A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion6. In contrast, the infarct area at 24 hours after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.

<table><tbody><tr><td>Buprenorphine (Buprenex)</td><td>Bedford Laboratories</td><td></td><td></td></tr><tr><td>Sodium Pentobarbital (Nembutal)</td><td>Ceva</td><td></td><td></td></tr><tr><td>Betadine</td><td>VWR internationals</td><td>200065-400</td><td></td></tr><tr><td>5 - 0 silk suture</td><td>Ethicon, Johnson &amp;amp; Johnson Medical</td><td>K890H</td><td></td></tr><tr><td>6 - 0 prolene suture&amp;nbsp;</td><td>Ethicon, Johnson &amp;amp; Johnson Medical</td><td>F1832</td><td></td></tr><tr><td>6 - 0 Ti- Cron suture</td><td>Ethicon, Johnson &amp;amp; Johnson Medical</td><td>F1823</td><td></td></tr><tr><td>Urethane&amp;nbsp;</td><td>Sigma</td><td>94300</td><td></td></tr><tr><td>Alconox</td><td>Alconox Inc.</td><td></td><td></td></tr><tr><td>Ventilator, MiniVent Model 845</td><td>Hugo Sachs</td><td>73-0043</td><td></td></tr><tr><td>Chest retractor or Thorax retractor</td><td>Kent Scientific corporation</td><td>INS600240</td><td>ALM Self-retaining, serrated, 7 cm long, 4 x 4 &quot;L&quot; shaped prongs, 3 mm x 3 mm</td></tr><tr><td>1.0 French Millar pressure catheter&amp;nbsp;</td><td>Millar Instruments&amp;nbsp;</td><td>SPR - 1000/NR</td><td></td></tr><tr><td>Powerlab</td><td>ADInstruments Pty Ltd.</td><td></td><td></td></tr><tr><td>LabChart&amp;reg; software</td><td>ADInstruments Pty Ltd.</td><td></td><td></td></tr><tr><td>Rectal probe</td><td>ADInstruments Pty Ltd.</td><td></td><td></td></tr></tbody></table>

permanent ligation of the left anterior descending coronary artery in mice: a model of post-myocardial infarction remodelling and heart failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

The extent of myocardial infarction can be assessed by Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) double staining. TTC is a redox indicator, which is converted to deep-red 1,3,5-triphenylformazan in living tissues due to the activity of various dehydrogenases in the presence of NADH8. Figure 1 shows a representative section of the heart at 24 hr after ligation of the left anterior descending coronary artery. Blue-stained areas indicate non-ischemic/normal regions. The myocardial area...

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Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

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Research

JoVE Journal

Medicine

27.7K Views.  Catholic University of Leuven. Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Video: Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...