1. 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

2. 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

3. 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

4. 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)

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

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

7. 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 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

8. 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

9. 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….

10. 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 Sep 1

11. 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 Sep 1

12. 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

13. 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]
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.

14. 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 Jul 23.

15. 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)

16. Body weight gain unaffected by surgery or treatment

17. Met enhanced progression to HF

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

19. Levo  early reversal of eccentric dilation index

20. %FS is consistent with treatment

21. 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

22. 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

23. 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

24. 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