Cardiovascular Research Mice and Rats are the most widely used - PowerPoint PPT Presentation

Rodents are commonly used in Cardiovascular Research • Mice and Rats are the most widely used species in cardiovascular research. • In the last 20 years, most papers reporting use of experimental models have used rodents, with the preponderance using mice.

Species use by year- Major CV Journals Mice Rat Dog Rabbit Circulation Research Circulation Hypertension ATVB Publications per year 150 150 125 175 150 125 125 100 125 100 100 75 100 75 75 75 50 50 50 50 25 25 25 25 0 0 0 0 1999 2009 2014 2018 1999 2009 2014 2018 1999 2009 2014 2018 1999 2009 2014 2018 Cardiovascular Research Eur. Heart J Nature Medicine AJP Heart and Circ Publications per year 150 25 100 150 125 125 20 75 100 100 15 75 50 75 10 50 50 25 5 25 25 0 0 0 0 1999 2009 2014 2018 1999 2009 2014 2018 1999 2009 2014 2018 2000 2009 2014 2018

Common uses for rodents in cardiovascular research Disease Rat Mouse Hypertension ++++ ++++ Atherosclerosis - ++++ Myocardial Infarction ++ ++ Heart Failure/hypertrophy ++ ++ Obesity/diabetes ++ ++ Other vascular Diseases • Aneurysms +++ • Injury models ++++ ++ • Stiffening and ++ ++ Fibrosis • Vasculitis +

Arrhythmia Research in Rodents I • Over the range of body mice form mice to whales, cardiac mass amounts 0.6% of body mass. • gross anatomy is remarkably similar. • comparable pacemaking and conduction structures. • Garrey postulated that a “critical mass,” is required to sustain reentrant arrhythmias (Garrey, 1924). • This led to belief that the mouse heart is “too small to fibrillate.” • Surface ECGs different • Mice have J wave without hypothermia • Action potentials different. Absent plateau in mice • TAC and ischemia lead to VT and atrial arrhythmias. • Trans-esophageal pacing used to non-invasively induce arrhythmias. • Perfused mouse hearts used for mapping, calcium imaging etc.

Arrhythmia Research in Rodents II • Mutations of PRKAG2, encoding gamma-2 subunit of the AMP-activated protein kinase (Blair et al., 2001; Gollob et al., 2001) leads to pre- excitation in humans and mice. • Several mutations lead to AV conduction abnormalities • Cx40 deficiency • Overexpression of human mutation of Nkx2-5 • Haploinsuffiency of transcription factor Tbx5, causes human Holt- Oram- like syndrome, (decreased Cx40 expression and AV block) • Deletion of calcium-activated potassium channel SK2 prolongs APD in atrial myocytes and leads to AF (Li et al., 2009).

Rats are widely used as models of hypertension and to study the genetics of hypertension • Spontaneously hypertensive rat vs WKY • Develop many aspect of human hypertension, including: • chronic renal disease • cardiac hypertrophy • Vascular dysfunction, fibrosis and rarefaction • outbred strains (SHR-SP) develop stroke at high rate • Dahl Salt sensitive vs. Salt resistant rat • Also develop severe renal lesions, proteinuria • Genetically modified • Both above models are used for gene segregation studies to identify hypertension causing alleles. • Drugs to treat hypertension in humans are effective in these models. Animal Models of Hypertension: A Scientific Statement From the American Heart Association Hypertension. 2019;73:e•••–e•••.

Additional Rat Models of Hypertension • Fawn-hooded hypertensive (FHH) rat • Milan hypertensive strain • Derived from Wystar Rats • Lyon hypertensive rat • Outbred Sprague Dawley Rats • Sabra hypertensive rat • Sensitive to DOCA-salt challenge • Genetically hypertensive rat • Inherited stress-induced arterial hypertension rat

Advantages of mice in cardiovascular studies • • Many Many labs have miniaturized methodology to accommodate mice studies. • Genetic modifications relatively easy and commonly used • Cell and organ targeted • Temporal control • Genetically altered lines readily available • Many reagents available including antibodies, PCR primers, cell isolation kits • Relatively inexpensive and thus possible to get adequate numbers • Genome well characterized

Mouse Models of Hypertension • Angiotensin II infusion • 140 ng/kg/min no increase in BP in normal mice • 300 ng/kg/min BP increase to 140 mmHg • 490 ng/kg/min BP increase to 165 mmHg • 1000 ng and above, no further increase above 490. Increased end-organ damage (aneurysms, • Kidney removal and salt sometimes added. • DOCA-salt hypertension • One kidney removed, DOCA pellet implanted and salt added to diet or water • BP increase to 145 mmHg • Diastolic dysfunction noted • L-NAME/high salt • Salt feeding (not in C57BL/6)

BP Measurement in Rodents

BP Measurement Variability and Uses

Response to low dose ang II

Transaortic constriction

Rodent myocardial infarction

Echocardiographic assessment of mouse cardiac function

Other echocardiographic measures • Vascular imaging • Flow velocity • Pulse wave velocity

Pulse wave velocity

Vascular Studies • Mesenteric • Carotid arteries • Aorta

Other imaging methods commonly used in rodents 18 F-FLT-PET/CT

Computerized tomography

Methods Commonly Used in Mice (Harrison lab) • Microinjection in brain nuclei • Renal denervation • Adoptive transfer of various immune cells • Flow cytometry of kidney, brain, vessels • Bone marrow transplant • Telemetry, tail cuff measures of blood pressure

Human monocyte-dependent T cell proliferation induced by high salt in vivo 10000 * #CD45 cells 8000 6000 CD4 Cells CD8 Cells 4000 2000 80 - 0 15 - CTRL HS 2000 * 40 - T cells 10 - #CD4 cells 1500 Monocytes (Label Cell 1000 Trace) 20 - 5 - 500 Normal 0 CTRL HS 0 - 0 - diet or 8000 salt-fed * Only T cells (no monocytes) #CD8 cells 6000 Control Diet 4000 High Salt Diet 2000 0 Analyze 12 days later CTRL HS

• What accomplishments/advancements have been made in the recent past (last 5 – 10 years) in cardiovascular disease through the use of rodents in research? • Numerous: Mechanisms of hypertension, heart remodeling, atherosclerosis, cardiovascular inflammation, vascular disease. • Could any of these accomplishments/advancements have been achieved in other species? • Possibly, but much slower, less likely • From your perspective, what is the likelihood of rodents replacing dogs as the preferred model for cardiovascular disease going forward? • This has already happened • What would be the tradeoffs in doing so? • See next slide • Would replacing dogs with another species compromise the quality or timelines of future advances? • See above. Dogs are not often used for cardiovascular research currently. Large animals clearly have some advantages.

Rodent Study Concerns • Hemodynamics • Heart rate 500 • Vascular Shear rates very different than humans (Suo et al, Arterioscler Thromb Vasc Biol. 2007;27:346-351) • BP remarkably similar across species • Phenotypes are sometimes not reproducible or stable • Genetic drift? • Microbiome? • Environment? • Differences in strains • Adaptation to genetic alterations • Specificity of genetic manipulations not always as suspected • Female/male differences don’t always recapitulate humans