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Articles in PresS. Am J Physiol Heart Circ Physiol (December 15, 2012). doi:10.1152/ajpheart.00674.2012
Asemu et al, H-00674-2012 Revision 2
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Enhanced Resistance to Permeability Transition in Interfibrillar Cardiac
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Mitochondria in Dogs: Effects of Aging and Long Term Aldosterone Infusion
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Girma Asemu1, Kelly A. O’Connell1, James W. Cox1, Erinne R. Dabkowski1, Wenhong
By:
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Xu1, Rogerio F. Ribeiro Jr1, Kadambari C. Shekar1, Peter A. Hecker1, Sharad Rastogi2,
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Hani N. Sabbah2, Charles L.Hoppel3 and William C. Stanley1
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From:
MD, USA
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Department of Medicine, Division of Cardiovascular Medicine, Henry Ford Hospital,
Detroit, MI, USA
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Division of Cardiology, Department of Medicine, University of Maryland, Baltimore,
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Departments of Pharmacology and Medicine, Case Western Reserve University School
of Medicine, Cleveland, OH 44106, USA
Running Title: Mitochondrial effects of aging and hypertrophy
Correspondence:
William C. Stanley, Ph.D.
Professor, Division of Cardiology, Department of Medicine
University of Maryland-Baltimore
20 Penn Street, HSF2, Room S-022
Baltimore, MD 21201
Phone: 410-706-3585
Fax: 410-706-3583
Email: [email protected]
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Copyright © 2012 by the American Physiological Society.
Asemu et al, H-00674-2012 Revision 2
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Abstract
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Functional differences between subsarcolemmal and interfibrillar cardiac mitochondria
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(SSM and IFM) have been observed with aging and pathological conditions in rodents. Results
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are contradictory and there is little information from large animal models. We assessed the
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respiratory function and resistance to mitochondrial permeability transition (MPT) in SSM and
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IFM from healthy young (1 year) and old (8 year) female beagles, and in old beagles with
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hypertension and LV wall thickening induced by 16 weeks of aldosterone infusion. MPT was
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assessed in SSM and IFM by Ca2+ retention and swelling. Healthy young and old beagles had
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similar mitochondrial structure, respiratory function and Ca2+-induced MPT within SSM and
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IFM subpopulations. On the other hand, oxidative capacity and resistance to Ca2+-induced MPT
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were significantly greater in IFM compared to SSM in all groups. Old beagles treated with
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aldosterone had greater LV wall thickness and worse diastolic filling, but normal LV chamber
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volume and systolic function. Treatment with aldosterone did not alter mitochondrial respiratory
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function, but accelerated Ca2+ -induced MPT in SSM, but not IFM, compared to healthy old and
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young beagles. In conclusion, in a large animal model oxidative capacity and resistance to MPT
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was greater in IFM than in SSM. Further, aldosterone infusion increased susceptibility to MPT
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in SSM, but not IFM. Together this suggests that SSM are less resilient to acute stress than IFM
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in the healthy heart, and are more susceptible to development of pathology with chronic stress.
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Keywords: Cardiac, diastolic dysfunction, heart failure, metabolism.
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Introduction
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Maintenance of normal membrane potential and respiratory function in cardiac
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mitochondria is critical for providing sufficient ATP supply for systolic and diastolic function
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and for prevention of cell death. In heart failure, there is an association between the degree of
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clinical severity and impaired mitochondrial oxidative capacity (42; 43; 51; 55).
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mitochondria are essential for energy transduction, they can also mediate myocardial pathology
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through mitochondrial permeability transition (MPT). MPT is a catastrophic event that occurs
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with the formation of a large pore that spans the inner and outer mitochondrial membranes,
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collapsing mitochondrial membrane potential and causing the mitochondria swelling and release
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cytochrome c and other matrix proteins that can trigger apoptosis (12; 39). MPT is triggered by
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cell stressors, particularly elevated [Ca2+], such as occurs with an acute bout of myocardial
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ischemia and reperfusion (12). Cardiac mitochondria from animals with advanced age or heart
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failure are more susceptible to stress-induced MPT (11; 12; 15; 17; 50), though the mechanism
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and impact of MPT in aging and heart failure are not clear.
While
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Heart muscle mitochondria are divided into two spatially distinct subpopulations:
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subsarcolemmal mitochondria (SSM) located in the outer region of the cell, and interfibrillar
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mitochondria (IFM) found between the myofibrils. Studies in rat myocardium found that IFM
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have a ~40% greater maximal rate of respiration per mg mitochondrial protein than SSM in
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normal rats (33; 34) or rats with infarct-induced heart failure (30; 38). Further, studies in rat
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heart found that IFM are more resistant to stress-induced MPT than SSM, as reflected in greater
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Ca2+ retention capacity and Ca2+-induced release of cytochrome c (15; 20; 35). This is not a
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consistent finding, as we found similar Ca2+ retention capacities in IFM and SSM in
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mitochondria from healthy hamsters and in normal rats or rats with infarct-induced heart failure
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(8; 9; 30). In contrast to rats and healthy hamsters, IFM from cardiomyopathic hamsters have
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lower respiration rates and a greater susceptibility to Ca2+-induced MPT than SSM (8; 16).
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Further, recent studies in diabetic mice suggest IFM have greater proteomic alterations and
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pathology than SSM (2; 7; 56). On the other hand, aging in rats had a greater impact on IFM
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than SSM, as seen in a decline in the ability of IFM to resist Ca2+-induced MPT, while this
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parameter does not change with age in SSM (15). There is little information regarding MPT in
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either young or old large animals. Young dogs with advanced heart failure with contractile
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dysfunction and left ventricular (LV) chamber enlargement have a greater susceptibility to MPT
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in permeabilized cardiomyocytes (50), but the effects of early stage heart failure associated with
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LV hypertrophy is not known. Further, differences between IFM and SSM resistance to MPT
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with age and heart failure have not been assessed.
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The goals of the present investigation were to assess Ca2+-induced MPT in IFM and SSM
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in healthy young and old dogs (1 and 8 years old), and in old dogs with hypertension and LV
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wall thickening induced by 16-weeks of aldosterone infusion.
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thickening and impaired diastolic filling, but did not increase end diastolic volume or pressure,
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nor impair contractile function. We hypothesized that IFM would have a greater resistance to
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Ca2+-induced MPT than SSM in young animals, and that IFM would have progressively
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increased susceptibility to MPT with age and with aldosterone infusion in older dogs. We
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studied females because they comprise approximately 60% of heart failure patients with
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preserved ejection fraction (4; 31). The size and structure of cardiac mitochondria change with
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advanced heart failure (44; 51) thus we assessed mitochondrial size and complexity in IFM and
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SSM using flow cytometry (6; 7; 36). In addition, we isolated mitochondria from the right and
This resulted in LV wall
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left ventricles, as there may be different responses to aging and aldosterone infusion between the
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two chambers.
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Methods
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Experimental Design.
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All procedures were conducted in accordance with the Guidelines for the Care and Use of
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Laboratory Animals (NIH publication No. 85-23), and were approved by the University of
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Maryland Institutional Animal Care and Use Committee. Three groups of female beagle dogs
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were studied: young untreated dogs (1 year old) (n=8), old untreated dogs (8 years old retired
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breeders) (n=8), and old dogs treated with an infusion of aldosterone for 16 weeks to induce
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hypertension (n=7). All dogs were purchased from a commercial vendor (Covance Research
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Products Inc, Cumberland, VA), and were ovariectomized by the vender two to six weeks prior
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to delivery. All of the terminal procedures and mitochondrial isolations were completed over a
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five week period and performed by the same personnel.
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mitochondria isolation and measurements were blinded to the group assignment.
The personnel performing the
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Dogs were housed two per pen and provided with food (Pedigree, Harlan Laboratories,
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Inc , Frederick, MD, USA) and water ad libitum. Following acclimatization to our facilities for 3
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to 5 days, arterial blood pressure was measured in the tail by oscillometric methods, and cardiac
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function was assessed by echocardiography as detailed below.
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Noninvasive Blood Pressure Monitoring
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Arterial blood pressure was measured in conscious dogs using an oscillometric automated
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blood pressure cuff (PetMAP) on the tail. The coccygeal artery was occluded by placing the cuff
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1cm distal to the base of the tail with arrow positioned along the ventral midline with the dog
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laying on its side. The values obtained for systolic and diastolic pressure and heart rate were
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taken from the average of 5 consecutive recordings, and mean arterial pressure (MAP) was
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calculated as diastolic pressure + (pulse pressure/3).
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Echocardiography
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LV function was evaluated by echocardiography (MyLab 30CV, Esaote North America,
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Inc, Indianapolis) using a 3.5-1.6 MHz probe. The echocardiographic examination was
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performed 7 to 3 days prior to the terminal study in all dogs. For the old dogs treated with
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aldosterone, measurements were also made 7 to 2 days prior to the pump instrumentation, and at
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15 weeks of aldosterone infusion. The exam was performed with the dog laying on the table in a
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right lateral decubitus position. The area was clipped free of hair and two-dimensional and M-
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mode echocardiography was performed with the probe on the left side. Images were obtained
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from a left parasternal approach at the mid–papillary muscle level and mitral value (46). No
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anesthesia or physical restraint was used. M-mode frames were recorded from the parasternal
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short axis, and PW Doppler measurements were recorded from the apical view. Anterior and
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posterior LV wall thicknesses were obtained at end diastole, and absolute wall thickness (AWT)
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was calculated as the sum of anterior and posterior wall thicknesses, and relative wall thickness
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(RWT) as AWT/end diastolic LV diameter. End systolic and diastolic volumes (ESV and EDV)
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were calculated as the LV diameter3 x 1.047 (46). Ejection fraction was calculated as (EDV -
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ESV) / EDV x 100. Transmitral Doppler indices peak rapid filling velocity (E) and peak atrial
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filling velocity (A) were measured and the E/A ratio was calculated.
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Venous blood was sampled in conscious dogs from a superficial forelimb 7 to 2 days
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prior to the terminal procedure. In the aldosterone treated dogs, blood was drawn prior to
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initiation of treatment and at 8 and 15 weeks of infusion. All blood samples were taken between
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11:00 and 14:00 in the nonfasted state.
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Infusion Pump Implantation
Aldosterone was continuously infusing with a battery powered programmable infusion
pump (iPRECIO model #MK02-V2, Data Sciences International, St. Paul, MN) that delivered
aldosterone into the jugular vein. D-aldosterone (Sigma Aldrich) was infused into the jugular
vein at a dose of 30 µg • kg-1 • day-1 in a solution of 15% ethanol, 50% DMSO, and 35% water at
a concentration of 10 mg of aldosterone/mL. Eight dogs initiated treatment with aldosterone, but
one dog was discontinued due to infection at the site of pump implantation, thus data are
reported for 7 animals. The young and old control animals were not instrumented. The dose of
aldosterone has been chosen based on our preliminary data showing that it increased blood
pressure. Pump implantation was performed under general anesthesia (propofol (4 to 6 mg/kg)
plus isoflurane (1.5- 3.0%) to effect), with local infiltration with bupivacaine (approximately 2
mL of 0.25%). The right external jugular vein was exposed and the pump cannula was inserted
and advanced 3-5 cm proximally. The vein was ligated distal to the point of insertion, and the
catheter secured to the vessel with suture ties. A subcutaneous pocket for the pump was created
lateral to the incision and the pump sutured in place, and the incision closed. Dogs were infused
at approximately 30 µL/day, with the exact rate adjusted to the body mass of the dog at the time
of implantation to deliver 30 µg • kg-1 • day-1. The pump reservoir was 900 µL and was refilled
percutaneously every 20-30 days through an injection port on the pump. The pump reservoir
was evacuated prior to refilling to insure the pump had properly discharged its contents, and was
then refilled using a 26 gauge needle. This procedure was done in conscious animals with no
evidence of discomfort. After 16 weeks, these dogs underwent a terminal procedure that was
identical to the young and old animals, as detailed below.
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Terminal Procedure to Assess Cardiac Function
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The terminal procedure was performed to assess left ventricular pressure and harvest the
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heart for mitochondrial studies. General anesthesia was induced as described above, and a 5-
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French manometer-tip catheter (Millar Instruments, Houston, TX) was inserted into the left
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carotid artery and advanced into the LV. Baseline LV pressure was recorded, and anesthesia was
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increased to 5% for 1 minute, and a left side thoracotomy was rapidly performed. The animal
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was euthanized by severing the superior vena cava, and the heart rapidly excised to obtain
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myocardial tissue samples. The total time from incision to removal of myocardial sample was
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<90 seconds. Myocardium for mitochondrial isolation was taken from the anterior free wall of
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the LV and RV (3 g and 1.5 g, respectively) and placed in ice cold buffer. Tissue from the lateral
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LV free wall was fixed in embedding medium (Tissue-Tek O.C.T. Compound, Sakura) for
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subsequent histological analysis and frozen at -80ºC. The residual LV and RV tissue was
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carefully dissected and weighed for assessment of LV and RV mass.
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Mitochondria Isolation and Measurements:
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Cardiac SSM and IFM were isolated using a protocol modified from Palmer et al. and
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Rosca et al (33; 43). Briefly, a ~3g transmural section of LV and 1.5 g of RV anterior free wall
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tissue were homogenized in a solution containing 100mM KCl, 50mM MOPS, 5.0mM MgSO4,
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1.0mM EGTA, 1.0mM ATP and 2mg/mL BSA (pH 7.4) without addition of collagenase, in
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contrast to our previous study in dogs where the minced muscle was treated with collagenase
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(43). IFM were released by treating the resuspended pellet with trypsin (5mg/g wet mass) in a
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similar solution as described above but without BSA, followed by mechanical homogenization
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(20; 43). Respiration was measured using glutamate+malate (20 and 10mM), pyruvate+malate
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(20 and 10 mM), palmitoylcarnitine (40uM), and succinate (20mM + 7.5µM rotenone) as
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substrates. State 4 was measured before and after addition of oligomycin (5ug/mL), and the
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respiratory control ratio was calculated as State 3/State 4 without oligomycin.
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Ca2+-induced MPT was evaluated in SSM and IFM from LV myocardium using two
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previously described methods (20-22). First, mitochondrial swelling was evaluated from the
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change in absorbance at 540 nm using a 96 well plate reader. Plates were read at 37°C and
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250ug/mL mitochondrial protein was added to each well in a calcium-free buffer (100mM KCl,
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50mM MOPS, 5mM KH2PO4, 1mM MgCl2, 5µM EGTA) with glutamate+malate as the
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substrate (5 and 2.55mM, respectively) and read for 20 minutes with readings taken every 7
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seconds.
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following the extramitochondrial [Ca2+] with a progressive exposure to Ca2+. Briefly, using a 96
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well fluorometric plate reader at 37°C, 250ug/mL of mitochondrial protein was suspended in the
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same calcium free buffer as previously mentioned with glutamate+malate as the substrate (5 and
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2.55mM).
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extramitochondrial [Ca2+] was recorded every 2 seconds using 750nM Calcium Green-5N.
Second, the capacity for Ca2+ uptake was evaluated in isolated mitochondria by
A bolus injection of 20nmol free Ca2+ was injected every 3 minutes, and
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Mitochondrial size and membrane potential were measured as previously described (6;
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36). Briefly, isolated SSM and IFM were stained with MitoTracker Green FM (Molecular
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Probes) and assessed using a flow cytometer (BD FACScan, BD Biosciences). The arithmetic
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mean output from the forward scatter detector was used as an index of mitochondrial size. For
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membrane potential, mitochondria were incubated with 5,5’,6,6’-tetrachloro-1,1’,3,3’-
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tetraethylbenzimidazol carbocyanine iodide (JC-1) (Molecular Probes, Carlsbad, CA) at a final
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concentration of 0.3 µM. The shift to orange is due to the dye forming aggregates upon
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polarization causing shifts in emitted light from 530 nm (green) to 590 nm (orange).
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Hydrogen peroxide production in isolated LV mitochondrial subpopulations was
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determined using the oxidation of the fluorogenic indicator amplex red in the presence of
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horseradish peroxidase. The concentrations of horseradish peroxidase and amplex red in the
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incubation were 0.1 unit/ml and 50 μM and detection of fluorescence was assessed on a
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Molecular Devices Flex Station 3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA)
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with 530 nm excitation and 590 nm emission wavelengths. Standard curves were obtained by
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adding known amounts of H2O2 to the assay medium in the presence of the substrates amplex red
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and horseradish peroxidase.
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glutamate/malate and succinate/rotenone as substrates.
H2O2 production was initiated in mitochondria using
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The activity of the citric acid cycle enzyme citrate synthase was measured in myocardial
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homogenates from frozen LV and RV samples at 37°C using a previously described
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spectrophotometric method (53). Histological analysis of LV samples for extracellular fibrosis,
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myocyte cross-sectional area and capillary density was assessed as previously described (45).
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Statistical Analysis. Values are shown as mean ± standard error. SSM and IFM values were
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compared using a 2-way repeated measures ANOVA with a Bonferroni post hoc test. A 1-way
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ANOVA was used for single parameters taken at the terminal time point. The time course for
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tail cuff blood pressure in the aldosterone treated dogs was compared to baseline using a
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repeated measures 1-way ANOVA. Echocardiographic data from baseline was compared to 15
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week values with a paired t-test. A p-value of <0.05 was considered significant. Statistical
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comparisons were not made between the LV and RV.
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RESULTS
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Effects of Aldosterone Infusion.
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Mean arterial pressure increased significantly compared to baseline, with a peak increase
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observed between 4-8 weeks of infusion of aldosterone followed by a gradual decline back to
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near baseline values by 15 weeks (Figure 1). No difference was observed in baseline serum
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aldosterone concentrations among the three groups (290 ±21, 235±28 and 272±37 pg/mL for the
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young, Old and Old+Aldo groups, respectively). Serum aldosterone concentration increased
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significantly at 8 weeks of aldosterone infusion to 423±54 pg/mL (P<0.001 vs. baseline values),
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but decreased to 79±21 pg/ml at 16 weeks infusion (P<0.05 compared to baseline and 8 weeks).
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This surprising finding suggests that chronic aldosterone infusion causes adaptations that
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increase the clearance of aldosterone from the circulation.
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Body mass was similar among the three groups (Table 1). Body mass in the Old+Aldo
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group increased by 1.6±0.4 kg from baseline to 16 week of aldosterone infusion (p<0.005)
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(Table 1). LV, RV and LV+RV mass were not significant different among groups, though there
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was a trend for a greater LV mass in the Old+Aldo.
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Tail cuff measurements acquired in conscious unrestrained dog 3 to 7 days prior to the
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terminal study show similar heart rate and blood pressure among groups (Table 2). Blood
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pressure in the untreated old and young dogs (Table 2) was not different from measurements
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taken at the same time point (4 to 10 days after arrival in our facility) in the group of old dogs
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that subsequently underwent infusion pump implantation and aldosterone infusion (systolic
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pressure 167±5 mmHg, diastolic pressure 88±5 mmHg, mean pressure 114±4 mmHg, and a heart
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rate of 113±9 beats/min). Invasive assessment of LV pressure in anesthetized animals showed
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no difference in heart rate, LV Peak systolic pressure, and maximum and minimum dP/dt. LV
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end diastolic pressure was significantly higher in the young dogs than in both the Old and
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Old+Aldo groups, though all dogs were in the normal healthy range (Table 2).
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Echocardiographic measurements showed no difference in LV chamber size and ejection fraction
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among groups, but increased LV wall thickness after 15 weeks of aldosterone infusion (Figure 2,
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Table 2).
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decrease in the peak E/A ratio in Old+Aldo dogs, however LV end diastolic pressure was normal
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(Figure 2 and Table 2). Histological assessment of LV myocardium found no increase in
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extracellular fibrosis or myocyte cross sectional area with aging or infusion of aldosterone (Table
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3). Further, capillary density was similar among the three groups. Taken together, while there
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was echocardiographic evidence for a modest decline in LV diastolic filling and wall thickening,
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there was no evidence for frank diastolic dysfunction or heart failure in the Old+Aldo group.
Diastolic dysfunction was detected via Doppler echocardiography as seen in a
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Mitochondrial Parameters
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Mitochondrial Yield and Size. The yields for SSM and IFM in young dogs was similar
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to values previously reported for LV myocardium from normal female mongrel dogs
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approximately 1.5 years of age (43). Total LV mitochondrial yield was significantly decreased
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in Old+Aldo group compared with Old and Young groups (Figure 3). Mitochondrial IFM yield
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was also lower in the Old+Aldo group compared with the Old group, and there was a significant
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decrease in mitochondria SSM yield in Old+Aldo compared with the Young group (Figure 3).
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In the RV, total mitochondrial yield was not significantly different among groups, except for a
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decrease in IFM yield in the Old+Aldo group compared with the Old group (Table 3).
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Myocardial activity of the Krebs cycle enzyme citrate synthase showed a significant decrease in
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activity in old dogs compared to young dogs, with no effect of aldosterone treatment, while in
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the RV there were no differences among groups (Table 3).
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Mitochondrial morphology was assessed by flow cytometry, and showed a main effect
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for larger mitochondrial size in SSM than IFM in both the LV and RV (Figure 3 and Table 4). In
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the LV, SSM were larger that IFM in the Old and Old+Aldo groups, but not in the Young group
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(Figure 3). The Old+Aldo group had larger SSM compared to the Young and Old groups,
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suggesting that aldosterone infusion caused selective mitochondrial enlargement in this
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subpopulation (Figure 3 and Table 4). Mitochondrial internal complexity was greater in SSM
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than IFM, and was significantly increased in SSM in the Old+Aldo group compared to the Old
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and Young groups in both the LV and RV. Mitochondrial membrane potential was lower in IFM
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than SSM in the Old and Old+Aldo groups in both the LV and RV, and was significantly
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increased in SSM in the Old+Aldo dogs compared to the Young groups (Figure 3 and Table 4).
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Mitochondrial Respiration.
State 3 respiration with glutamate+malate, pyruvate+malate,
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palmitoylcarnitine, and succinate+rotenone was not affected either by age or treatment in IFM or
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SSM in the LV or RV (Figure 4 and Table 4). There was a significantly higher state 3
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respiration rate in IFM than SSM with all substrates in the LV (Figure 4). A similar effect was
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observed in the RV for glutamate+malate and palmitoylcarnitine, but not for pyruvate+malate or
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succinate+rotenone.
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The respiratory control ratio (RCR) was not different between SSM and IFM except for a
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small but significantly greater RCR for IFM with succinate+rotenone as a main effect (Figure 4,
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right panel). The RCR was lower in the Old+Aldo group compared to the Old group with
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succinate+rotenone,
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pyruvate+malate
and
was
strongly
trending
lower
with
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palmitoylcarnitine (p<0.051), suggesting that aldosterone treatment lowered mitochondrial
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responsiveness to ADP stimulation of respiration (Figure 4).
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LV state 4 respiration measured in the absence of oligomycin was higher in IFM than
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SSM with all substrates, and was not different among the three groups with pyruvate+malate,
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palmitoylcarnitine, and succinate+rotenone as substrates (Figure 5, left panels). With
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glutamate+malate as substrate, there was a higher state 3 rate in Young and Old+Aldo than in the
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Old group in IFM, but no differences among groups in SSM. State 4 was also measured with the
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addition of oligomycin to block ATP production by Complex V and to thus provide a measure of
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proton leak across the inner mitochondrial membrane. This resulted in lower rates of respiration
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(Figure 5, right panel) with persistently higher state 4 rates in IFM than SSM, but no difference
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among groups. In the RV, state 4 measured in the absence of oligomycin was higher in IFM than
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SSM with all substrates except pyruvate+malate (Table 4), and the Old+Aldo group had a higher
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state 4 rate than the Young and Old groups. State 4 was lowered by the addition of oligomycin,
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with higher rates in IFM with glutamate+malate and palmitoylcarnitine as substrates (Table 4),
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left panel). There were no differences among groups with the exception of higher rates in IFM
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with glutamate+malate in Old+Aldo in the IFM group (Table 4).
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Permeability Transition. Two established methods were used to assess Ca2+-induced MPT in
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LV mitochondria. First, MPT was assess from mitochondrial swelling induced by high [Ca2+] as
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reflected by the decrease in absorbance at 540 nm following the addition of a bolus of Ca2+ to
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isolated mitochondria. Measurement of initial baseline absorbance prior to the addition of Ca2+
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showed greater absorbance in IFM than SSM in all groups, and higher absorbance in the
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Old+Aldo group in both SSM and IFM compared to the young and old groups (Figure 6). There
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was a decrease in absorbance with addition of either 0.5 or 1.0 μmoles Ca2+/mg protein in all
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groups, with a greater decline in IFM than SSM, and no differences among the three groups with
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SSM or IFM (Figure 7).
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Ca2+-induced MPT also was assessed from the measurement of the ability of isolated
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mitochondria to take up added Ca2+. IFM had significantly enhanced Ca2+ retention capacity
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compared to SSM, as reflected by significantly lower extramitochondrial [Ca2+] for a given
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cumulative Ca2+ load (Figure 8). There was a significantly greater sensitivity to Ca2+-induced
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MPT in SSM from the Old+Aldo group compared to the Old and Young groups, as reflected by
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a higher extramitochondrial [Ca2+] for a given cumulative Ca2+ load (Figure 9). There were no
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significant differences in this relationship in IFM among the three groups (Figure 9). These
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results indicate that IFM were more resistant to Ca2+-induced MPT than SSM, and that
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aldosterone infusion caused a significant increase in susceptibility to MPT only in the SSM.
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Hydrogen Peroxide Production.
The capacity for mitochondrial generation of H2O2 was
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assessed in SSM and IFM from LV myocardium using the amplex red assay. There were no
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differences among groups within mitochondrial subpopulations with either glutamate+malate or
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succinate in the presence of the rotenone to inhibit complex I (Table 5). The maximal rate
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measured with succinate plus rotenone showed no difference between IFM and SSM. With
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glutamate+malate as substrates, the rate was lower in IFM than SSM as a main effect and within
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the Young and Old groups (Table 5).
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Discussion.
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The present investigation used a large animal model to assess the function and structure
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of the spatially distinct subpopulations of cardiac mitochondria under normal conditions in
327
young and old females, and with aldosterone infusion that induced transient hypertension,
328
modest LV wall thickening and impaired LV filling. There are three main findings from this
329
study. First, we found that respiratory capacity and resistance to Ca2+-induced MPT were
330
enhanced in IFM compared to SSM in LV myocardium in both young and old dogs (15; 35).
331
Second, we observed that aldosterone infusion in old dogs reduced the yield and increased the
332
size of SSM, but not IFM. Third, aldosterone infusion decreased the capacity for Ca2+ uptake in
333
SSM, but had no effect on IFM. Thus, in the healthy female heart, mitochondria located in the
334
outer region of the cell are more susceptible to permeability transition and have a lower capacity
335
for ATP generation. Further, in response to aldosterone infusion, SSM became enlarged and less
336
resistant to stress, while the mitochondria found among the myofibrils appear unchanged.
337
We found that the rate of oxidative phosphorylation with pyruvate+malate,
338
palmitoylcarnitine and succinate+rotenone was approximately 30-50% greater in IFM than SSM
339
in a large animal. This finding was evident in both young and old dogs and with aldosterone
340
infusion, and in both RV and LV myocardium. In a previous study in young male mongrel dogs,
341
we did not observe any differences in state 3 or state 4 respiration between IFM and SSM in
342
healthy animals or in dogs with chronic heart failure due to irreversible injury caused by multiple
343
micro-infarctions (43). This suggests that enhanced respiration in IFM from the canine heart may
344
be unique to females. Sex differences between IFM and SSM have not be reported, however a
345
previous study that examined only SSM found that both young (~4 months) or mature (>32
346
months) female rabbits had a significantly higher state 3 rate with either complex I or II
18
Asemu et al, H-00674-2012 Revision 2
19
347
substrates compared to males.
On the other hand, SSM from adult mice showed no sex
348
differences in state 3 or 4 with pyruvate+malate as substrate (47). Thus at present it is unclear if
349
the greater respiratory capacity in IFM than SSM that we observed in female dogs is present in
350
males.
351
The greater resistance to stress-induced permeability transition in IFM compared to SSM
352
is consistent with previous results from young and old healthy rats (15; 20; 35). Hoppel et al
353
showed that isolated IFM from rat heart had a 50% greater Ca2+-uptake capacity than SSM.
354
Further, SSM released cytochrome c and matrix enzymes in response to added Ca2+, while IFM
355
were relatively unaffected (35).
356
retention capacities in IFM and SSM in normal rats or rats with infarct-induced heart failure, and
357
in healthy hamsters (8; 30). Hofer et al found that IFM from senescent rats are more susceptible
358
to Ca2+-induced MPT, while this parameter was unaffected by age in SSM (15). We did not
359
observe any effect of age in indices of Ca2+-induced MPT in the present study, however 8 year
360
old beagles should be considered middle aged, as the typical lifespan of this strain is 13.5±0.2
361
years (24). A previous study found that healthy 11±2 year old male beagles had a decrease in
362
succinate dehydrogenase activity in myocardial tissue homogenates and a qualitative increased
363
vacuolization of mitochondria by visual analysis of electron micrographs compared to 4±1 year
364
old animals, however mitochondrial respiration and MPT were not assessed (3). Future studies
365
should evaluate these parameters in senescent dogs, where greater mitochondrial dysfunction
366
would be expected.
367
On the other hand, we previously observed similar Ca2+
The effects of LV hypertrophy and heart failure on MPT in humans or large animal
368
models are not well understood.
Young dogs with advanced heart failure due to
369
microembolizations have a greater susceptibility to MPT in permeabilized cardiomyocytes (50),
19
Asemu et al, H-00674-2012 Revision 2
20
370
but the effects of early stage heart failure associated with LV hypertrophy has not been report in
371
humans or large animals. Recent studies found that atrial mitochondria from middle aged
372
diabetic patients without heart failure are more sensitive to Ca2+-induced MPT and have greater
373
production of H2O2 than mitochondrial from nondiabetic patients (1). We found IFM from
374
cardiomyopathic hamsters had greater susceptibility to Ca2+-induced MPT than normal healthy
375
hamster, while SSM were similar (8). The implications of these findings are not clear, as the role
376
of MPT in normal cardiac physiology and the progression of heart failure is unresolved and
377
remains under extensive investigation.
378
The underlying mechanisms responsible for the differences in respiration and resistance
379
to MPT between IFM and SSM are not known. High resolution scanning electron microscopy
380
analysis revealed clear structural differences between the two subpopulations in rat
381
cardiomyocytes, with SSM having more lamelliform and less tubular cristae than IFM (40),
382
which could affect function.
383
respiration and MPT (54), however there are no differences between IFM and SSM in
384
phospholipid composition, cardiolipin molecular species, or phospholipid fatty acid composition
385
in normal or heart failure dogs or rats (20; 30; 41).
386
sphingolipid composition varies tremendously between IFM and SSM in rat heart, particularly
387
for long chain ceramides (26).
388
susceptibility to permeability transition, the role of these compounds in mitochondrial
389
physiology are not yet clear, thus it is premature to postulate a mechanistic link to the present
390
findings (26). It is also possible that differences in key proteins involved in MPT pore structure
391
or regulation differ between subpopulations, however there was no difference in protein levels
392
for the voltage dependent anion channel, cyclophilin D, or the adenine nucleotide translocator
Mitochondrial phospholipid composition can clearly impact
On the other hand, mitochondrial
While this could impact mitochondrial respiration or
20
Asemu et al, H-00674-2012 Revision 2
21
393
between SSM and IFM in young or old rat hearts (15). Clearly additional work is needed to
394
elucidate the underlying reasons for the distinctions between the two subpopulations.
395
A novel finding of the present investigation is that IFM from the RV exhibited enhanced
396
respiratory capacity relative to SSM, consistent with current and previous findings from LV
397
mitochondria (30; 33; 34; 38). While the functional and metabolic differences between the two
398
chambers have been described (23; 48; 49), little is known about differences in mitochondrial
399
structure and function. Direct comparisons of the LV and RV in healthy young male rats found a
400
20% greater mitochondrial volume density in the RV as assessed by electron microscopy, but no
401
difference in mitochondrial respiratory capacity in permeablized cardiomyocyte bundles or in the
402
activity of complexes I-IV or citrate synthase in myocardial homogenates (29). In contrast,
403
mitochondrial volume density was not different between LV and RV myocardium in healthy
404
young pigs (52). Here we show that mitochondrial yield and functional differences between
405
SSM and IFM were qualitatively similar in the LV and RV, with responses to aging and
406
aldosterone infusion (Figure 3-8). The present investigation was not designed to statistically
407
compare the RV with the LV, as this would require a 3-way ANOVA (chamber x mitochondrial
408
population x age/treatment group) and thus a much larger sampled size to achieve acceptable
409
statistical power. We did not assess Ca2+-induced MPT in RV mitochondria, which could have
410
been differentially effected by aldosterone compared to LV mitochondria. It has recently been
411
proposed that mitochondrial dysfunction in the RV plays a key role in the pathological
412
adaptations to pulmonary hypertension, and that mitochondrial targeted interventions may be
413
therapeutic (37). Additional work focused on interventricular differences in the mitochondrial
414
response to left and right side failure is warranted.
21
Asemu et al, H-00674-2012 Revision 2
22
415
In the present investigation, we aimed to develop a new model of pathological LV
416
hypertrophy in dogs using a continuous infusion of aldosterone with an implanted motorized
417
infusion pump. This general approach has been used in young unilaterally nephrectomized rats
418
to induce hypertrophic heart failure with mitochondrial dysfunction (19), but to our knowledge
419
has not been attempted in dogs. Our goal was to mimic key aspects of heart failure with
420
preserved ejection fraction, thus we used old animals as they are more susceptible to myocardial
421
pathology in response to stress (18; 28). Females were used rather than males because they
422
make up approximately 55-65% of heart failure patients with preserved ejection fraction (4; 31).
423
Further, dogs were ovariectomized to eliminate the confounding effects of ovulation and to better
424
reflect conditions in postmenopausal women. We wished to avoid the confounding effects of
425
thoracic or renal surgery required for aortic constriction, nephrectomy or the Page renal wrap
426
procedure (13; 14; 32), thus we used a controlled intravenous infusion of aldosterone via a pump
427
implanted in the neck. Previous studies showed that aldosterone at 12 to 15 µg · kg-1 · day-1
428
increased mean arterial pressure by ~15 mmHg for up to 10 days in young mongrel dogs (5; 10;
429
27). We aimed to increase mean pressure by 15 to 20 mmHg, and in dose escalating pilot studies
430
in our model system we found that this required approximately 30 µg · kg-1 · day-1. While we
431
clearly accomplished the desired increase in serum aldosterone and blood pressure out to 8
432
weeks (Figure 1), we were surprised to see a fall in both parameters by 15 weeks of infusion. In
433
hindsight, a progressive increase in the aldosterone infusion rate would likely have maintained
434
hypertension and resulted in clear pathological LVH. Further, simultaneous infusion of other
435
potent vasoconstrictor compounds with toxic myocardial effects, such as angiotensin II or
436
endothelin, should be considered to further accelerate the development of hypertension, LVH
22
Asemu et al, H-00674-2012 Revision 2
23
437
and heart failure. Further investigation is needed with a more severe and prolonged hormonal
438
stress to elicit advanced heart failure with preserved ejection fraction.
439
A limitation of our experimental design is the lack of a control group implanted with an
440
infusion pump and treated with vehicle for 16 weeks. This group was not included for ethical
441
reasons. Blood pressure transiently increased following initiation of aldosterone infusion (Figure
442
1), suggesting that this was due to aldosterone infusion. However, in the absence of a parallel
443
vehicle treated group, it is not possible to definitively attribute the observed differences in blood
444
pressure and other parameters to aldosterone infusion.
445
A hallmark of heart failure with preserved ejection fraction is impair diastolic filling as
446
reflected by delayed mitral inflow (e.g. decrease in the E/A ratio) and elevated LV end diastolic
447
pressure. In the present investigation we observed a modest but significant decrease in the E/A
448
ratio but no increase in LV end diastolic pressure. In contrast, Mathieu et al observed a similar
449
modest but significant decrease in the E/A ratio (from 1.51 to 1.22), but also an increase in LV
450
end diastolic pressure from 14 to 23 mmHg in beagles two month after myocardial infarction
451
induced by coronary ligation (25). This suggests that these modest reductions in the E/A should
452
be interpreted with caution, as they clearly do not necessarily correspond with changes LV filling
453
pressure.
454
In conclusion, we demonstrate in a large animal model that resistance to MPT is greater
455
in IFM than in SSM in young and old female dogs.
When old dogs were stressed with
456
aldosterone infusion, there was selective enlargement of SSM and greater susceptibility to MPT,
457
with no change to IFM. Further, we observed a greater capacity for oxidative phosphorylation in
458
IFM than SSM in all three groups. Together these findings show that in a large animal model
459
there are clear differences between cardiac mitochondrial subpopulations under normal
23
Asemu et al, H-00674-2012 Revision 2
24
460
conditions, and response to 15 weeks of aldosterone infusion. The implications of this finding to
461
more advanced LVH and heart failure require further study.
462
463
24
Asemu et al, H-00674-2012 Revision 2
464
465
Acknowledgments
This work was supported by the National Institutes of Health grant numbers HL074237 and
466
HL110731.
25
467
468
Disclosures
469
None to disclose.
470
471
25
Asemu et al, H-00674-2012 Revision 2
472
473
474
26
Reference List
475
476
1. Anderson EJ, Rodriguez E, Anderson CA, Thayne K, Chitwood WR and Kypson AP.
477
Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-
478
dependent pathways. Am J Physiol Heart Circ Physiol 300: H118-H124, 2011.
479
2. Baseler WA, Dabkowski ER, Williamson CL, Croston TL, Thapa D, Powell MJ,
480
Razunguzwa TT and Hollander JM. Proteomic alterations of distinct mitochondrial
481
subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction. Am
482
J Physiol Regul Integr Comp Physiol 300: R186-R200, 2011.
483
3. Bhashyam S, Parikh P, Bolukoglu H, Shannon AH, Porter JH, Shen YT and Shannon
484
RP. Aging is associated with myocardial insulin resistance and mitochondrial dysfunction.
485
Am J Physiol Heart Circ Physiol 293: H3063-H3071, 2007.
486
4. Bhatia RS, Tu JV, Lee DS, Austin PC, Fang J, Haouzi A, Gong Y and Liu PP.
487
Outcome of heart failure with preserved ejection fraction in a population-based study. N
488
Engl J Med 355: 260-269, 2006.
489
5. Cowley AW, Jr., Skelton MM and Merrill DC. Are hypertensive effects of aldosterone,
490
angiotensin, vasopressin, and norepinephrine chronically additive? Hypertension 8: 332-
491
343, 1986.
26
Asemu et al, H-00674-2012 Revision 2
27
492
6. Dabkowski ER, Baseler WA, Williamson CL, Powell M, Razunguzwa TT, Frisbee JC
493
and Hollander JM. Mitochondrial dysfunction in the type 2 diabetic heart is associated
494
with alterations in spatially distinct mitochondrial proteomes. Am J Physiol Heart Circ
495
Physiol 299: H529-H540, 2010.
496
7. Dabkowski ER, Williamson CL, Bukowski VC, Chapman RS, Leonard SS, Peer CJ,
497
Callery PS and Hollander JM. Diabetic cardiomyopathy-associated dysfunction in
498
spatially distinct mitochondrial subpopulations. Am J Physiol Heart Circ Physiol 296:
499
H359-H369, 2009.
500
8. Galvao TF, Brown BH, Hecker PA, O'Connell KA, O'shea KM, Sabbah HN, Rastogi
501
S, Daneault C, des RC and Stanley WC. High intake of saturated fat, but not
502
polyunsaturated fat, improves survival in heart failure despite persistent mitochondrial
503
defects. Cardiovasc Res 93: 24-32, 2012.
504
9. Galvao TF, Khairallah RJ, Dabkowski ER, Brown BH, Hecker PA, O'Connell KA,
505
O'shea KM, Sabbah HN, Rastogi S, Daneault C, des RC and Stanley WC. Marine n3
506
Polyunsaturated Fatty Acids Enhance Resistance to Mitochondrial Permeability Transition
507
in Heart Failure, but Do Not Improve Survival. Am J Physiol Heart Circ Physiol 2012.
508
10. Gonzalez-Campoy JM, Kachelski J, Burnett JC, Jr., Romero JC, Granger JP and
509
Knox FG. Proximal tubule response in aldosterone escape. Am J Physiol 256: R86-R90,
510
1989.
27
Asemu et al, H-00674-2012 Revision 2
511
512
513
514
11. Gustafsson AB and Gottlieb RA. Heart mitochondria: gates of life and death. Cardiovasc
Res 77: 334-343, 2008.
12. Halestrap AP and Pasdois P. The role of the mitochondrial permeability transition pore in
heart disease. Biochim Biophys Acta 1787: 1402-1405, 2009.
515
13. Hart CY, Meyer DM, Tazelaar HD, Grande JP, Burnett JC, Jr., Housmans PR and
516
Redfield MM. Load versus humoral activation in the genesis of early hypertensive heart
517
disease. Circulation 104: 215-220, 2001.
518
14. Hittinger L, Shannon RP, Bishop SP, Gelpi RJ and Vatner SF. Subendomyocardial
519
exhaustion of blood flow reserve and increased fibrosis in conscious dogs with heart
520
failure. Circ Res 65: 971-980, 1989.
521
15. Hofer T, Servais S, Seo AY, Marzetti E, Hiona A, Upadhyay SJ, Wohlgemuth SE and
522
Leeuwenburgh C. Bioenergetics and permeability transition pore opening in heart
523
subsarcolemmal and interfibrillar mitochondria: effects of aging and lifelong calorie
524
restriction. Mech Ageing Dev 130: 297-307, 2009.
525
28
16. Hoppel CL, Tandler B, Parland W, Turkaly JS and Albers LD. Hamster
526
cardiomyopathy. A defect in oxidative phosphorylation in the cardiac interfibrillar
527
mitochondria. J Biol Chem 257: 1540-1548, 1982.
28
Asemu et al, H-00674-2012 Revision 2
528
17. Javadov S and Karmazyn M. Mitochondrial permeability transition pore opening as an
529
endpoint to initiate cell death and as a putative target for cardioprotection. Cell Physiol
530
Biochem 20: 1-22, 2007.
531
29
18. Jugdutt BI, Jelani A, Palaniyappan A, Idikio H, Uweira RE, Menon V and Jugdutt
532
CE. Aging-related early changes in markers of ventricular and matrix remodeling after
533
reperfused ST-segment elevation myocardial infarction in the canine model. effect of early
534
therapy with an angiotensin II type 1 receptor blocker. Circulation 122: 341-351, 2010.
535
19. Kamalov G, Deshmukh PA, Baburyan NY, Gandhi MS, Johnson PL, Ahokas RA,
536
Bhattacharya SK, Sun Y, Gerling IC and Weber KT. Coupled calcium and zinc
537
dyshomeostasis and oxidative stress in cardiac myocytes and mitochondria of rats with
538
chronic aldosteronism. J Cardiovasc Pharmacol 53: 414-423, 2009.
539
20. Khairallah RJ, O'shea KM, Brown BH, Khanna N, des RC and Stanley WC.
540
Treatment with docosahexaenoic acid, but not eicosapentaenoic acid, delays Ca2+-induced
541
mitochondria permeability transition in normal and hypertrophied myocardium. J
542
Pharmacol Exp Ther 335: 155-162, 2010.
543
21. Khairallah RJ, Sparagna GC, Khanna N, O'shea KM, Hecker PA, Kristian T, Fiskum
544
G, des RC, Polster BM and Stanley WC. Dietary supplementation with docosahexaenoic
545
acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid
546
fatty acid composition and prevents permeability transition. Biochim Biophys Acta 1797:
547
1555-1562, 2010.
29
Asemu et al, H-00674-2012 Revision 2
30
548
22. Khairallah RJ, Kim J, O'Shea KM, O'Connell KA, BBH, Galvao TDRC, Polster BM,
549
Hoppel CL and Stanley WC. Improved mitochondrial function with diet-induced increase
550
in either docosahexaenoic acid or arachidonic acid in membrane phospholipids . PLoS One
551
7: e34402, 2012.
552
23. Kusachi S, Nishiyama O, Yasuhara K, Saito D, Haraoka S and Nagashima H. Right
553
and left ventricular oxygen metabolism in open-chest dogs. Am J Physiol 243: H761-H766,
554
1982.
555
556
557
24. Lloyd RD, Taylor GN and Miller SC. Does low dose internal radiation increase lifespan?
Health Phys 86: 629-632, 2004.
25. Mathieu M, El OB, Touihri K, Hadad I, Mahmoudabady M, Thoma P, Metens T,
558
Bartunek J, Heyndrickx GR, Brimioulle S, Naeije R and Mc EK. Ventricular-arterial
559
uncoupling in heart failure with preserved ejection fraction after myocardial infarction in
560
dogs - invasive versus echocardiographic evaluation. BMC Cardiovasc Disord 10: 32,
561
2010.
562
26. Monette JS, Gomez LA, Moreau RF, Bemer BA, Taylor AW and Hagen TM.
563
Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.
564
Biochem Biophys Res Commun 398: 272-277, 2010.
565
566
27. Montani JP, Mizelle HL, Adair TH and Guyton AC. Regulation of cardiac output
during aldosterone-induced hypertension. J Hypertens Suppl 7: S206-S207, 1989.
30
Asemu et al, H-00674-2012 Revision 2
567
28. Munagala VK, Hart CY, Burnett JC, Jr., Meyer DM and Redfield MM. Ventricular
568
structure and function in aged dogs with renal hypertension: a model of experimental
569
diastolic heart failure. Circulation 111: 1128-1135, 2005.
570
29. Nouette-Gaulain K, Malgat M, Rocher C, Savineau JP, Marthan R, Mazat JP and
571
Sztark F. Time course of differential mitochondrial energy metabolism adaptation to
572
chronic hypoxia in right and left ventricles. Cardiovasc Res 66: 132-140, 2005.
573
31
30. O'shea KM, Khairallah RJ, Sparagna GC, Xu W, Hecker PA, Robillard-Frayne I, des
574
RC, Kristian T, Murphy RC, Fiskum G and Stanley WC. Dietary omega-3 fatty acids
575
alter cardiac mitochondrial phospholipid composition and delay Ca2+-induced
576
permeability transition. J Mol Cell Cardiol 47: 819-827, 2009.
577
31. Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL and Redfield MM. Trends
578
in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med
579
355: 251-259, 2006.
580
581
32. Page IH. A METHOD FOR PRODUCING PERSISTENT HYPERTENSION BY
CELLOPHANE. Science 89: 273-274, 1939.
582
33. Palmer JW, Tandler B and Hoppel CL. Biochemical properties of subsarcolemmal and
583
interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252: 8731-8739,
584
1977.
31
Asemu et al, H-00674-2012 Revision 2
585
34. Palmer JW, Tandler B and Hoppel CL. Biochemical differences between
586
subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of
587
procedural manipulations. Arch Biochem Biophys 236: 691-702, 1985.
588
589
590
32
35. Palmer JW, Tandler B and Hoppel CL. Heterogeneous response of subsarcolemmal
heart mitochondria to calcium. Am J Physiol 250: H741-H748, 1986.
36. Papanicolaou KN, Ngoh GA, Dabkowski ER, O'Connell KA, Ribeiro RF, Stanley WC
591
and Walsh K. Cardiomyocyte deletion of mitofusin-1 leads to mitochondrial
592
fragmentation and improves tolerance to ROS-induced mitochondrial dysfunction and cell
593
death. Am J Physiol Heart Circ Physiol 302: H167-H179, 2012.
594
595
596
37. Piao L, Marsboom G and Archer SL. Mitochondrial metabolic adaptation in right
ventricular hypertrophy and failure. J Mol Med (Berl) 88: 1011-1020, 2010.
38. Rennison JH, McElfresh TA, Okere IC, Vazquez EJ, Patel HV, Foster AB, Patel KK,
597
Chen Q, Hoit BD, Tserng KY, Hassan MO, Hoppel CL and Chandler MP. High-fat
598
diet postinfarction enhances mitochondrial function and does not exacerbate left ventricular
599
dysfunction. Am J Physiol Heart Circ Physiol 292: H1498-H1506, 2007.
600
601
39. Ricchelli F, Sileikyte J and Bernardi P. Shedding light on the mitochondrial permeability
transition. Biochim Biophys Acta 1807: 482-490, 2011.
32
Asemu et al, H-00674-2012 Revision 2
33
602
40. Riva A, Tandler B, Loffredo F, Vazquez E and Hoppel C. Structural differences in two
603
biochemically defined populations of cardiac mitochondria. Am J Physiol Heart Circ
604
Physiol 289: H868-H872, 2005.
605
41. Rosca M, Minkler P and Hoppel CL. Cardiac mitochondria in heart failure: Normal
606
cardiolipin profile and increased threonine phosphorylation of complex IV. Biochim
607
Biophys Acta 1807: 1373-1382, 2011.
608
609
610
42. Rosca MG and Hoppel CL. Mitochondria in heart failure. Cardiovasc Res 88: 40-50,
2010.
43. Rosca MG, Vazquez EC, Kerner J, Parland W, Chandler MP, Stanley WC, Sabbah
611
HN and Hoppel CL. Cardiac mitochondria in coronary microembolization-induced heart
612
failure: decrease in respirasomes and oxidative phosphorylation. Cardiovasc Res 80: 30-39,
613
2008.
614
44. Sabbah HN, Sharov V, Riddle JM, Kono T, Lesch M and Goldstein S. Mitochondrial
615
abnormalities in myocardium of dogs with chronic heart failure. J Mol Cell Cardiol 24:
616
1333-1347, 1992.
617
45. Sabbah HN, Stanley WC, Sharov VG, Mishima T, Tanimura M, Benedict CR, Hegde
618
S and Goldstein S. Effects of dopamine beta-hydroxylase inhibition with nepicastat on the
619
progression of left ventricular dysfunction and remodeling in dogs with chronic heart
620
failure. Circulation 102: 1990-1995, 2000.
33
Asemu et al, H-00674-2012 Revision 2
621
46. Sahn DJ, DeMaria A, Kisslo J and Weyman A. Recommendations regarding
622
quantitation in M-mode echocardiography: results of a survey of echocardiographic
623
measurements. Circulation 58: 1072-1083, 1978.
624
47. Sanz A, Hiona A, Kujoth GC, Seo AY, Hofer T, Kouwenhoven E, Kalani R, Prolla
625
TA, Barja G and Leeuwenburgh C. Evaluation of sex differences on mitochondrial
626
bioenergetics and apoptosis in mice. Exp Gerontol 42: 173-182, 2007.
627
48. Schwartz GG, Greyson CR, Wisneski JA, Garcia J and Steinman S. Relation among
628
regional O2 consumption, high-energy phosphates, and substrate uptake in porcine right
629
ventricle. Am J Physiol 266: H521-H530, 1994.
630
49. Schwartz GG, Steinman S, Garcia J, Greyson C, Massie B and Weiner MW.
631
Energetics of acute pressure overload of the porcine right ventricle. In vivo 31P nuclear
632
magnetic resonance. J Clin Invest 89: 909-918, 1992.
633
34
50. Sharov VG, Todor AV, Imai M and Sabbah HN. Inhibition of mitochondrial
634
permeability transition pores by cyclosporine A improves cytochrome C oxidase function
635
and increases rate of ATP synthesis in failing cardiomyocytes. Heart Fail Rev 10: 305-310,
636
2005.
637
51. Sharov VG, Todor AV, Silverman N, Goldstein S and Sabbah HN. Abnormal
638
mitochondrial respiration in failed human myocardium. J Mol Cell Cardiol 32: 2361-2367,
639
2000.
34
Asemu et al, H-00674-2012 Revision 2
640
641
52. Singh S, White FC and Bloor CM. Myocardial morphometric characteristics in swine.
Circ Res 49: 434-441, 1981.
642
53. Srere PA. Citrate synthase. Methods Enzymol 13: 3-5, 1969.
643
54. Stanley WC, Khairallah RJ and Dabkowski ER. Update on lipids and mitochondrial
644
function: impact of dietary n-3 polyunsaturated fatty acids. Curr Opin Clin Nutr Metab
645
Care 15: 122-126, 2012.
646
647
648
35
55. Stanley WC, Recchia FA and Lopaschuk GD. Myocardial substrate metabolism in the
normal and failing heart. Physiol Rev 85: 1093-1129, 2005.
56. Williamson CL, Dabkowski ER, Baseler WA, Croston TL, Alway SE and Hollander
649
JM. Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of
650
subcellular spatial location. Am J Physiol Heart Circ Physiol 298: H633-H642, 2010.
651
652
35
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653
654
655
Table 1. Body and heart mass.
Young
Old
Old+ALDO
1.0±0.02
7.8±0.1*
7.9±0.1*
-
-
10.4±0.6
Terminal Body Mass (kg)
10.2±0.3
11.2±0.5
12.0±0.8§
LV mass (g)
50.0±1.7
52.4±3.5
61.2±4.7
RV mass(g)
18.3±1.1
18.6±1.6
19.6±1.3
LV mass + RV mass (g)
68.3±2.4
70.9±5.0
80.8±5.6
LV mass/terminal body mass (g/kg)
4.91±0.18
4.66±0.22
5.11±0.02
RV mass/body mass
1.79±0.11
1.65±0.10
1.66±0.10
Age at Termination (years)
Initial Body Mass (kg)
656
657
* p<0.0001 Compared to Young
658
§ p<0.005 Compared to initial body mass within Old+Aldo group by paired t-test
659
† p<0.02 Compared to Old LV mass/terminal body mass
660
# p<0.02 Compared to Young LV mass/terminal body mass
661
662
663
36
Asemu et al, H-00674-2012 Revision 2
664
665
666
37
Table 2. Cardiovascular function. Tail cuff and echocardiography measurements were made in
conscious unrestrained dog 3 to 7 days prior to the terminal study. LV pressure was assessed by
catheterization in anesthetized animals immediately before euthanasia.
667
Young
Old
Old+ALDO
112±9
151±4
82±3
105±2
110±4
156±7
85±3
109±3
119±7
174±12
95±8
121±9
Echocardiographic Measurements
EDD (mm)
ESD (mm)
FS (%)
EDV (mL)
ESV (mL)
LV ejection fraction (%)
Posterior Wall Thickness (mm)
Anterior Wall Thickness (mm)
Absolute Wall Thickness (mm)
Relative Wall Thickness
E/A ratio
22.2±1.2
13.3±0.5
0.38±0.03
12.1±1.9
2.7±0.8
75.4±3.9
10.2±0.6
6.5±0.3
16.7±0.8
0.95±0.09
1.28±0.04
24.6±0.9
15.1±0.8
0.39±0.02
16.0±1.6
3.8±0.6
76.6±2.3
9.9±0.6
7.1±0.4
17.1±1.0
0.81±0.03
1.25±0.03
25.9±1.5
16.4±0.9#
0.39±0.03
19.2±3.7
4.2±0.8
76.6±3.8
12.0±0.4†
8.7±0.6#
20.7±0.7#†
0.81±0.05
1.00±0.04#†
Left Ventricle Pressure
Heart Rate (beats/min)
LV peak systolic pressure (mmHg)
LV end diastolic pressure (mmHg)
LV maximum dP/dt (mmHg/s)
LV minimum dP/dt (mmHg/s)
129±7
113±5
7.0±0.9
1956±173
-2367±381
113±5
102±6
3.7±0.4#
1740±196
-1834±185
114±7
92±5#
4.0±0.5#
1472±79
-1773±182
Tail Cuff Measurements
Heart Rate (beats/min)
Systolic Blood Pressure (mmHg)
Diastolic Blood Pressure (mmHg)
Mean Blood Pressure (mmHg)
668
669
† p<0.05 Compared to Old, # p<0.05 Compared to Young
670
671
37
Asemu et al, H-00674-2012 Revision 2
672
38
Table 3. Activity of citrate synthase in the LV and RV, and histological analysis of LV myocardium. .
673
Citrate Synthase Activity (µmols · g-1 · min-1)
LV
RV
Young
Old
Old+ALDO
135±10
105±6
98±8*
92±9
104±9
120±9
529±17
9.4±0.5
2410±132
529±16
9.4±0.7
2067±94
546±28
10.0±0.6
2106±92
Histological Analysis of LV Myocardium
Myocyte cross sectional area (μm2)
Volume fraction of interstitial fibrosis (%)
Capillary density (capillaries/mm2)
674
675
* p<0.05 Compared to Young
676
677
678
38
Asemu et al, H-00674-2012 Revision 2
679
680
681
39
Table 4. LV mitochondrial H2O2 production with succinate+rotenone or glutamate+malate. Values are
expressed as nmoles · mg mito prot-1 · min-1.
Young
Old
Old+ALDO
Succinate+Rotenone -SSM
128±8
120±8
132±8
Succinate+Rotenone -IFM
127±10
130±8
135±15
Glutamate+Malate -SSM
62.8±10.5
69.6±8.1
54.5±9.4
Glutamate+Malate IFM
46.5±13.3*
37.7±4.7 *
45.9±7.8
Main Effect
vs. SSM
0.001
682
683
*p<0.05 IFM compared to SSM
684
685
39
Asemu et al, H-00674-2012 Revision 2
686
Figure Legends
687
Figure 1. Time course of arterial blood pressure in the old dogs treated with aldosterone.
688
*p<0.05 compared to baseline, † p<0.01compared to baseline.
40
689
690
Figure 2. Echocardiographic assessment of LV function in the old dogs treated with aldosterone.
691
Measurements were made prior to treatment and after 15 weeks of aldosterone infusion.
692
693
Figure 3. LV mitochondrial yield and flow cytometry results. *p<0.05 IFM compared to SSM,
694
† p<0.05 compared to Old within given mitochondrial subpopulation, # p<0.05 compared to
695
Young within given mitochondrial subpopulation.
696
697
Figure 4. RV mitochondrial yield and flow cytometry results. *p<0.05 IFM compared to SSM, †
698
p<0.05 compared to Old within given mitochondrial subpopulation, # p<0.05 compared to
699
Young within given mitochondrial subpopulation.
700
701
Figure 5. LV mitochondrial State 3 respiration in in SSM (white bars) and IFM (black bars) (left
702
panel) and the respiratory exchange ratio (RCR; State 3/State 4 without oligomycin) (right
703
panel). *p<0.05 IFM compared to SSM.
704
705
Figure 6. RV mitochondrial State 3 respiration in in SSM (white bars) and IFM (black bars) (left
706
panel) and the respiratory exchange ratio (RCR; State 3/State 4 without oligomycin) (right
707
panel). *p<0.05 IFM compared to SSM, # p<0.05 compared to Young within given
708
mitochondrial subpopulation.
40
Asemu et al, H-00674-2012 Revision 2
41
709
710
Figure 7. LVmtochondrial State 4 respiration in SSM (white bars) and IFM (black bars) without
711
oligomycin (left panel) and with oligomycin to inhibit ATP production by complex V. *p<0.05
712
IFM compared to SSM, † p<0.05 Compared to Old,
713
714
Figure 8. RV mitochondrial State 4 respiration in SSM (white bars) and IFM (black bars)
715
without oligomycin (left panel) and with oligomycin to inhibit ATP production by complex V.
716
*p<0.05 IFM compared to SSM, † p<0.05 compared to Old within given mitochondrial
717
subpopulation, # p<0.05 compared to Young within given mitochondrial subpopulation.
718
719
Figure 9. Baseline absorbance at 540 nm of LV mitochondria without addition of Ca2+. *p<0.05
720
IFM compared to SSM, † p<0.05 Compared to Old,
721
722
Figure 10. Change in absorbance at 540 nm, index of LV mitochondrial swelling, following
723
addition of either 0.5 or 1.0 μmoles Ca2+/mg mitochondrial protein. *p<0.05 IFM compared to
724
SSM.
725
726
Figure 11. Comparison between IFM and SSM from the LV for each group of dogs for
727
extramitochondrial Ca2+ concentration plotted as a function of the cumulative Ca2+ load.
728
*p<0.05 IFM compared to SSM.
729
730
41
Asemu et al, H-00674-2012 Revision 2
42
731
Figure 12. Extramitochondrial Ca2+ concentration plotted as a function of cumulative Ca2+ load
732
for SSM (upper panel) and IFM (Lower Panel) from the LV. *p<0.05 compared to Young, †
733
p<0.05 compared to Old.
734
735
736
737
738
42
Asemu et al, H-00674-2012 Revision 2
43
739
43
Figure 1
Mean Artterial Pressu
ure (mmHg)
150
140
130
†
*
*
†
*
120
110
P<0.002 vs. baseline for main effect
100
4
6
8
10
12
14
Baseline 2
Duration of Aldosterone Infusion (weeks)
Figure 2
30
LV EDD (mm)
25
100
Ejection Fraction (%)
80
20
0.8
40
0.4
20
5
Pretreatment
+ Aldo
Ald
15 weeks
Posterior Wall
Thickness (mm)
P<0 002
P<0.002
12
0
14
10
8
8
6
6
4
4
2
2
Pretreatment
+ Aldo
15 weeks
Pretreatment
+ Aldo
Ald
15 weeks
Anterior Wall
Thickness (mm)
0
0.0
Pretreatment
+ Aldo
Ald
15 weeks
Posterior + Anterior
Thickness (mm)
25
P<0.002
12
10
0
P<0.0003
60
10
14
E/A Ratio
1.2
15
0
1.6
20
N.S.
15
10
5
Pretreatment
+ Aldo
15 weeks
0
Pretreatment
+ Aldo
15 weeks
Figure 3
Left Ventricular Mitochondria
#†
15
#
#†
*
*
200
100
Young
Old
SSM
P<0.0001 main effect
IFM vs SSM
80
IFM
*
#†
8
*
40
20
Young
g
0
Old+Aldo
60
0
IFM
300
Old
Old+Aldo
(JC-1 aggre
egate/monomer)
100
(Arbittrary Units)
*
5
0
Mitochond
drial Complexity
*
10
SSM
P<0.0001 main effect
IFM vs SSM
400
Membrane potential ΔΨm
(mg prot./g wet wt.)
Mitochondrial Y
Yield
IFM vs SSM
20
500
(Arbitrary Un
nits)
SSM Yield
IFM Yield
Total Yield
Mitochondria
al Size
25 P<0.004 main effect
Young
Old Old+Aldo
SSM
IFM
P<0 0001 main effect
P<0.0001
IFM vs SSM
6
*
#
*
4
2
0
Youngg
Old
Old+Aldo
Figure 4
Right Ventricular Mitochondria
SSM Yield
IFM Yield
Total Yield
5
Old
P<0.007 main effect
IFM vs SSM
Old+Aldo
#†
*
(Arbittrary Units)
80
60
20
0
Young
g
Old
Old+Aldo
*
*†
*
200
8
Y Data
40
P<0.001 main effect
IFM vs SSM
IFM
300
0
(JC-1 aggre
egate/monomer)
100
Young
SSM
100
Membrane potential ΔΨm
0
Mitochond
drial Complexity
*
10
400
(Arbitrary Units)
15
500
Mitochondrial Sizze
M
P<0.03 main effect
IFM vs SSM
20
((mg prot./g wet wt.)
Mitochondrial Yielld
M
25
Young
Old Old+Aldo
P<0.001 main effect
IFM vs SSM
6
*
*
4
2
0
Youngg
Old
Old+Aldo
Left Ventricular Mitochondria
State 3 (nA O mg-1 min-1)
P<0.03 main effect SSM vs IFM
*
200
100
0
Young
Old
IFM
SSM
Glutamate + Malate
300
Old+Aldo
Respiratory Excha
ange Ratio
Figure 5
6
4
2
0
*
200
*
*
150
100
50
0
Young
Old
Old+Aldo
100
500
400
Young
Old
Old+Aldo
4
2
1
0
*
*
200
100
Young
Old
Young
Old
Old+Aldo
6
5
4
3
2
1
0
Young
Old
Old+Aldo
Succinate + Rotenone
P<0.001 main effect SSM vs IFM
*
P<0.002
main effect vs Old
3
Succinate + Rotenone
300
0
Respiratory Exchange R
Ratio
*
Old+Aldo
Respiratory
y Exchange Ratio
State 3 (nA O mg-1 m
min-1)
State 3 (nA O mg-1 min-1)
*
200
0
Old+Aldo
Palmitoylcarnitine
P<0.001 main effect SSM vs IFM
*
Old
5
Palmitoylcarnitine
300
Young
Pyruvate + Malate
P<0.001 main effect SSM vs IFM
Respirato
ory Exchange Ratio
State 3 (nA O mg-1 min-1)
Pyruvate + Malate
250
Glutamate + Malate
8
3.0 P<0.03 main effect SSM vs IFM
P<0.02
main effect vs Old
2.5
2.0
1.5
1.0
0.5
0.0
Young
Old
Old+Aldo
Right Ventricular Mitochondria
SSM
State 3 (nA O mg-1 min-1)
Glutamate + Malate
350 P<0.001 main effect SSM vs IFM
300
*
250
200
150
100
50
0
Young
Old
Old+Aldo
IFM
Respiratory Exchan
nge Ratio
Figure 6
10
8
6
4
2
0
200
150
100
50
Old
Old+Aldo
Respirato
ory Exchange Ratio
250
Young
State 3 (nA O mg-1 m
min-1)
300
P<0.001 main effect SSM vs IFM
*
250
*
*
150
100
50
Young
Old
Old+Aldo
8
3
2
1
0
200
100
Old
Old
Old+Aldo
4
2
0
Old+Aldo
Young
Old
Old+Aldo
Succinate + Rotenone
Respiratory Exchange Ratio
State 3 (nA O mg-1 min-1)
300
Young
Young
6
Succinate + Rotenone
400
0
Old+Aldo
Palmitoylcarnitine
200
0
Old
4
Palmitoylcarnitine
350
Young
Pyruvate + Malate
5
Respiratory Exchange R
Ratio
State 3 (nA O mg-1 min-1)
Pyruvate + Malate
300
0
Glutamate + Malate
12
3.0
2.5
#
2.0
*
1.5
1.0
0.5
0.0
Young
Old
Old+Aldo
Left Ventricular Mitochondria
Figure 7
State 4 (nA O mg
g-1 min-1)
No Oligomycin
70
60
*
State 4 (nA O mg-1 min-1)
State 4 (nA O mg-11 min-1)
†
70
60
30
20
20
10
10
Young
Old
Old+Aldo
Pyruvate + Malate
P<0.001 main effect SSM vs IFM
0
80
60
60
40
40
20
20
0
80
Young
Old
Old+Aldo
Palmitoylcarnitine
P<0.001 main effect SSM vs IFM
*
60
*
*
80
40
20
180
0
Young
Young
Old
Old+Aldo
Succinate + Rotenone
P<0.01 main effect SSM vs IFM
0
180
120
60
60
Young
Old
Old
Old+Aldo
Pyruvate + Malate
P<0.001 main effect SSM vs IFM
*
Young
*
*
Old
Old+Aldo
Palmitoylcarnitine
P<0.001 main effect SSM vs IFM
*
20
120
0
*
*
60
40
0
P<0.001 main effect SSM vs IFM
40
30
0
+ Oligomycin
Glutamate + Malate
50
*
40
80
State 4 (nA O mg-1 min-1)
*†
P<0.001 main effect SSM vs IFM
50
IFM
SSM
Glutamate + Malate
Old+Aldo
0
Young
*
*
Old
Old+Aldo
Succinate + Rotenone
P<0.001 main effect SSM vs IFM
*
Young
*
Old
*
Old+Aldo
Right Ventricular Mitochondria
Figure 8
No Oligomycin
SSM
State 4 (nA O mg-1 m
min-1)
Glutamate + Malate
60
60
P<0.004 main effect SSM vs IFM
*
40
IFM
#†
20
0
20
Young
g
Old
0
Old+Aldo
State 4 (n
nA O mg-1 min-1)
90
*
P<0.001 main effect SSM vs IFM
*
*
60
30
0
80
60
Old
Old+Aldo
*
60
*
*
30
Young
Old
0
Old+Aldo
P<0.001 main effect SSM vs IFM
*
*
20
Old
Old+Aldo
Succinate + Rotenone
180 P<0.005 main effect SSM vs IFM
*
120
*
P<0.001 main effect SSM vs IFM
0
180
*
*
Young
Old
Old+Aldo
Succinate + Rotenone
P<0.001 main effect SSM vs IFM
120
*
60
Old
Old+Aldo
#†
60
Young
Old
60
20
Young
Young
Palmitoylcarnitine
80
40
0
Young
g
90 P<0.001 main effect SSM vs IFM
40
0
*
Pyruvate + Malate
Palmitoylcarnitine
State 4 (nA O mg-1 min-1)
P<0.001 main effect SSM vs IFM
40
Pyruvate + Malate
State 4 (nA
A O mg-1 min-1)
+ Oligomycin
Glutamate + Malate
Old+Aldo
0
Young
*
*
Old
Old+Aldo
Figure 9
0.5
Absorbance
0.4
SSM
*
IFM
*
#
*# †
0.3
0.2
0.1
0.0
Young
Old
Old+Aldo
ce (AU)
Relative Absorbanc
Figure 10
Young
1.1
1.0
** ** *
+ 0.5 µmols
0.9
200
600
800
Young
1.1
****
1.0
1
0
0.9
Old
******
SSM
IFM
*****
*
+ 1.0 µmols Ca2+
0.8
0.8
0
200
400
600
800
1000 1200
Old
1.1
******
1.0
1
0
***
200
400
600
800
Time (secs)
1000 1200
0
200
400
600
800
1000 1200
Old+ Aldo
** * ** *
10
1.0
0.9
+ 1.0 µmols Ca2+
* **
+ 0.5 µmols Ca2+
1.1
0.8
0
*** ** *
1.0
0.9
0.9
0.8
**
0.9 + 0.5 µmols Ca2+
1000 1200
Old+ Aldo
1.1
**
Ca2+
400
1.1
1.0
0.8
0
R
Relative Absorbance (AU)
SSM
IFM
****
+ 1.0 µmols Ca2+
0.8
0
200
400
600
800
1000 1200
Time (secs)
0
200
400
600
800
Time (secs)
1000 1200
Extramitochondriall [Ca2+] (AU)
Extramitochondrial [C
Ca2+] (AU)
Extra
amitochondrial [Ca2++] (AU)
Figure 11
Young
4000
SSM
IFM
3000
2000
**
1000
0
0
4000
50
100
150
*
*
200
*
*
250
Old
3000
2000
**
*
*
*
*
300
*
1000
0
4000
0
50
100
150
200
Old+ Aldo
3000
2000
250
*
*
*
**
300
**
1000
0
0
50
100
150
200
250
300
Cumulative Ca2+ Load (nmols/mg mito. prot.)
Extramitochondria
al [Ca2+] (AU)
Extramitochondrial [Ca2+] (A
AU)
Figure 12
SSM
4000
3000
Young
Old
Old+Aldo
2000
†
†
*
*
*
*
†
*
†
*
†
*
1000
0
0
2000
1500
50
100
Young
Old
Old+Aldo
150
200
250
300
IFM
1000
500
No Significant Differences
0
0
100
200
300
Cumulative Ca2+ Load (nmols/mg mito. prot.)