Structural and functional remodeling following pharmacologic intervention in volume overload heart failure Kristin Lewis, DVM Pathology Resident/Graduate Research Associate The Ohio State University, Columbus, OH The Research Institute, Nationwide Children’s Hospital, Columbus, OH

Why are we interested in heart failure? ~5 million Americans currently have CHF ~550,000 new cases diagnosed annually Contributes to ~300,000 deaths each year Sudden death is 6-9x more likely in CHF patients than in the general population HF is responsible for >11 million physician visits annually and more hospitalizations than all forms of cancer combined http://www.emoryhealthcare.org/heart-failure/learn-about-heart-failure/statistics.html

2 types of hemodynamic overload  HF Pressure Overload Volume Overload Two types of hemodynamic load have been characterized that lead to HF. Both are different with respect to structural and functional effects. Increased afterload Concentric hypertrophy Fibrosis Examples: Hypertension Aortic stenosis Increased preload Eccentric hypertrophy ECM degradation Examples: Aortic/Mitral regurgitation Area opposite infarct Ventricular septal defect

Progression of Volume Overload (VO) to Heart Failure Reversible Irreversible Valvular Dysfunction Aortic regurgitation Mitral regurgitation Systolic Dysfunction Volume Overload Septal Defects Diastolic Dysfunction HF Death Over the course of years, valvular disease, septal defects and infarcts result in volume overload. Initially, there is adaptive, reversible LV remodeling and LV dysfunction. In our opinion, this is the optimal time to begin treatment. If left untreated, patients progress to irreversible, decompensated heart failure. At this stage, treatment is designed to reduce clinical signs. Even with treatment, patients can ultimately die. Unfortunately, treatment is often delayed until this irreversible stage. Little is understood about the progression of volume overload. We do know the timeline. What is known is that it is caused by VD, SD, MI. …leading to VO, which induces dysfunction and if left untreated leads to overt hf and death. The remodeling process takes years, but once you are in overt heart failure there is a limitation to the clinical corrections that can be made. Current clinical treatment is focused during the later stages of HF following signs of dysfunction. IN MOST CASES Therapy is USUSALLY WHEN patients are in HF; past the point of no return!!!. Our goal is to understand the remodeling process better, therefore lead to earlier detection by diagnostic markers and find new therapeutic options for treatment within THE WINDOW OF OPPORTUNITY. The best opportunity is to focus on the reversible stage. SEGWAY. Drugs are here, we know know that we can reverse the remodeling surgically, but a pharm reversal would be ideal. Is there evidence that that type of strategy could work. The answer is yes. NEXT SLIDE. Myocardial Infarct LV Remodeling LV Dysfunction Overt HF Time (months to years) Time (months)

Overall hypothesis: Early intervention will result in return of LV structure and function to baseline levels

Volume overload-induced HF with aortocaval fistula (ACF) in the rat 18g Aorta

ACF progressive increase in LVDd Sham 4 wk ACF LVDd LVDs 8 wk ACF M-mode echo: Top of photo = anterior wall (or cranial wall) Bottom of photo = posterior wall (or caudal wall) 4 week ACF: Early compensated HF: increased dilation, decreased systolic function, normal diastolic function Roughly equivalent to NYHA stage 1-2. Few to no clinical signs 8 week ACF: Late compensated HF: further increased dilation, further decreased systolic function, normal diastolic function Roughly equivalent to NYHA stage 3. Clinical signs at exertion 15 week ACF: 1) Decompensated HF: 3+ dilation, 3+ systolic dysfunction, 2+ diastolic dysfunction 2) Roughly equivalent to NYHA stage 4. Clinical signs at rest. 15 wk ACF

VO is accompanied by functional deterioration % FS * *= P < 0.05 vs. Sham * Fractional shortening (FS) is the fraction of any diastolic dimension that is lost in systole. When referring to endocardial luminal distances, it is EDD minus ESD divided by EDD (times 100 when measured in percentage). Fractional shortening underestimates dysfunction, esp since there is increased preload and decreased afterload in this model. PV loops are a load-independent measure of function. Stroke volume: SV=EDV-ESV CO=HR*SV PV loops: Lower left corner: Mitral valve opens and ventricle begins filling Lower right corner: mitral valve closes = end diastolic volume = represents preload Top right: Aortic valve opens = beginning of systole Top left: Aortic valve closes = end systolic volume Yellow line = ESPVR (end systolic pressure volume relationship) = the maximum pressure that the ventricle is capable of generating at any given volume Right PV loops (Systolic failure PV loops): -Decreased ESPVR slope indicating reduced inotropy -Dilated ventricle (i.e. eccentric hypertrophy) --Increased compliance (i.e. decreased EDPVR slope) -Increased ESV and EDV  decreased SV and EF

Will reversal of ACF improve LV structure and function? Stent graft Suture State that it is PE190 Make a better PE tube for pic. TAKE IMAGE AND PLACE A RULER BY IT. State that we have shift in hemodynamic load here….

LV chamber geometry is normalized 4wks post-reversal Question: Will Reversal of ACF Improve LV Structure and Function? We looked at LVDd which was sig enlarged in ACF at 4 weeks *= P < 0.05 vs. Sham †= P < 0.05 vs. ACF Hutchinson KR, et al. J Appl Physiol. 2011 Sep 1

ACF reversal  decreased LV contractility @ 4 weeks & normalization of LV contractility @ 11 weeks Sham ACF Only ACF + Reversal * * 4 wk ACF ± 4 wk Rev Pressure (mmHg) † * Current surgical management of volume overload-induced heart failure (HF) leads to variable recovery of left ventricular (LV) function despite a return of LV geometry. End-systolic elastance (Ees), the slope parameter of the end-systolic pressure (ESP)-volume (ESV) relation (ESPVR) There are multiple components to the ESPVR. These are slope and shift. Our results show us that at 4 weeks following reversal we see decreased contractile function in Reversal. This suggests that from the period of 4 to 8 weeks we still have systolic dysfunction that does not return until and 15 weeks. The importance of this is that under stress the rats could have inadequate cardiac function resulting in ischemia and infarct. In order to id systolic function regardless of volume we use load independent measures, which is obtained using a venous occlusion. Need to focus more one why a shift to the right is evidence of a decrease in contractility. SLOWLY walk through the volume shift vs. the slope. For first pv loop talk about what parts of the loop are. 4 wk ACF ± 11 wk Rev *= P < 0.05 vs. Sham Volume (µL) †= P < 0.05 vs. ACF Hutchinson KR, et al. J Appl Physiol. 2011 Sep 1

In a rat model of ACF-induced volume overload: AIM 1 In a rat model of ACF-induced volume overload: Determine the optimal time to initiate medical therapy by comparing the temporal efficacy of β-blocker (metoprolol) or myofilament Ca2+ sensitizer (levosimendan) therapy

Beta-blocker: Metoprolol Preferentially binds to β1-AR in the heart & blocks NE binding Clinical mechanism of action poorly understood: Theoretically:  HR, contractility, conduction velocity, relaxation rate Clinically:  contractility Benefit may be 2o to blockade of excess Epi/NE stimulation Beta blockers…wikipedia There are three known types of beta receptor, designated β1, β2 and β3 receptors.[9] β1-adrenergic receptors are located mainly in the heart and in the kidneys.[8] β2-adrenergic receptors are located mainly in the lungs, gastrointestinal tract, liver, uterus, vascular smooth muscle, and skeletal muscle.[8] β3-adrenergic receptors are located in fat cells.[10] http://www.cvpharmacology.com/cardioinhibitory/beta-blockers.htm Beta-adrenoceptors are coupled to a Gs-proteins, which activate adenylyl cyclase to form cAMP from ATP. Increased cAMP activates a cAMP-dependent protein kinase (PK-A) that phosphorylates L-type calcium channels, which causes increased calcium entry into the cell. Increased calcium entry during action potentials leads to enhanced release of calcium by the sarcoplasmic reticulum in the heart; these actions increase inotropy (contractility). Gs-protein activation also increases heart rate (chronotropy). PK-A also phosphorylates sites on the sarcoplasmic reticulum, which lead to enhanced release of calcium through the ryanodine receptors (ryanodine-sensitive, calcium-release channels) associated with the sarcoplasmic reticulum. This provides more calcium for binding the troponin-C, which enhances inotropy. Finally, PK-A can phosphorylate myosin light chains, which may contribute to the positive inotropic effect of beta-adrenoceptor stimulation. http://www.cvpharmacology.com/cardioinhibitory/beta-blockers.htm

Levosimendan (and OR-1896) act through multiple cardiovascular targets For example, the increase in the amplitude of the intracellular Ca2+ transient – in response to the activation of the β-adrenergic – cAMP – protein kinase A signaling pathway – augments force production through an increase in the Ca2+ saturation of cTnC. This manner of myocardial force augmentation is associated with a significant increase in myocardial oxygen demand, which is a limit to the pharmacological utilization of the β-adrenergic signaling pathway in the diseased heart. 3 mechanisms: Positive inotropy: Cellular target: Cardiomyocytes Subcellular target: Myofilaments Molecular target: Calcium-saturated form of troponin C The consequence of levosimendan binding is that the Ca2+-saturated cTnC is stabilized in the presence of the drug this conformational change involves a prolonged interaction between cTnC and cardiac troponin I, thereby promoting contractile force in the presence of levosimendan without an increase in the amplitude of intracellular Ca2+ transient. Molecular mechanism: Calcium sensitization Vasodilation: Cellular target: Vascular smooth muscle cells Vasodilation during levosimendan administration has been demonstrated at the arterial sides of the pulmonary, coronary, and peripheral circulations, and at the venous sides of the portal and saphenous Subcellular target: Sarcolemma Molecular target: ATP-sensitive K+ channels Molecular mechanism: Hyperpolarization Cardioprotection: Subcellular target: Mitochondria Molecular mechanism: Protection of mitochondria in ischemia-reperfusion Papp Z, et al. Int J Cardiol. 2011 Jul 23.

Study Design Sprague dawley rats, 210-260 g Treatment: Vehicle: water Metoprolol: 30 mg/kg x 4 wk, 50 mg/kg x 4 wk, 80 mg/kg x 3 wk Levosimendan: 1 mg/kg 0 wk Treatment start Hemodynamics Necropsy 4 wk (n=10) (n=8) ACF SHAM 15 wk VEH MET LEVO ECHO (q2w) (n=9)

Body weight gain unaffected by surgery or treatment

Met enhanced progression to HF

Levo & Met  delayed and enhanced increases in LVDd, respectively

Levo  early reversal of eccentric dilation index

%FS is consistent with treatment

Summary In our model of volume overload: Metoprolol accelerates the progression to HF Levosimendan delays the progression to HF Treatment started at lower LVDd 1) return to pre-surgical LVDd 2) maintenance of LVDd

Next steps Current study: Future studies: Structure: Function: ECHO Routine histology, organ weights Collagen content, TGF-β MMPs/TIMPs α-MHC, β-MHC Function: PV Loops ANP, BNP, Connexin 43 Future studies: Repeat current study + myocyte isolation ACF + earlier treatment ACF + reversal + treatment

Next steps Current study: Future studies: In vivo: Ex vivo: ECHO PV loops Ex vivo: Organ weights/ratios Routine histology: heart, liver, lungs, kidney Picrosirius red qPCR: Col1a1, Col3a1, elastin, α-MHC, β-MHC, ANP, BNP, TGF-β Immunoblot: MMP-13, MT1-MMP, MMP-7, MMP-9, TIMP-2 Future studies: Repeat current study + myocyte isolation ACF + earlier treatment ACF + reversal + treatment

Acknowledgements Nationwide Childrens The Ohio State University Lucchesi lab Pam Lucchesi Anu Guggilam Maarten Galanctowicz Aaron Trask Kathryn Halleck Kirk Hutchinson Aaron West Mary Cismowski Jean Zhang Vivarium Natalie Snyder Veterinary Biosciences Funding Sources ACVP/STP Coalition Fellowship NIH HL056046 Nationwide Children’s Hospital