Larry V Hryshko

Hôpital St-Boniface Hospital, Winnipeg, Manitoba, Canada

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Publications (61)252.37 Total impact

  • The Journal of Heart and Lung Transplantation 04/2015; 34(4):S148-S149. · 5.61 Impact Factor
  • The Journal of Heart and Lung Transplantation 04/2015; 34(4):S269-S270. · 5.61 Impact Factor
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    ABSTRACT: Background Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unutilized donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability prior to transplantation. We sought to evaluate the impact of different oxygen carriers on the preservation of myocardial function during EVHP. Methods Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours, and were transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, N=6), whole blood (RBC+Plasma, N=6), an acellular hemoglobin based oxygen carrier (HBOC, N=8), or HBOC plus plasma (HBOC+Plasma, N=7) were added to STEEN solution to achieve a perfusate hemoglobin concentration of 40 g/L. Results Perfusate composition impacted the preservation of systolic (T5 dP/dtmax: RBC+Plasma=903±99, RBC=771±77, HBOC+Plasma=691±82, HBOC=563±52 mmHg/second, p=0.047) and diastolic (T5 dP/dtmin: RBC+Plasma=-574±48, RBC=-492±63, HBOC+Plasma=-326±32, HBOC=-268±22 mmHg/second, p<0.001) function, and the development of myocardial edema (Weight gain: RBC+Plasma=6.6±0.9, RBC=6.6±1.2, HBOC+Plasma=9.8±1.7, HBOC=16.3±1.9 grams/hour, p<0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (Injury score: RBC+Plasma=0.0±0.0, RBC=0.8±0.3, HBOC+Plasma=2.6±0.2, HBOC=1.75±0.4, p<0.001) and less troponin-I release (Troponin-I fold change T1-T5: RBC+Plasma=7.0±1.7, RBC=13.1±1.6, HBOC+Plasma=20.5±1.1, HBOC=16.7±5.8, p<0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (Oxidized phosphatidylcholine compound fold change T1-T5: RBC+Plasma=1.83±0.20 vs. RBC=2.31±0.20, p<0.001; HBOC+Plasma=1.23±0.17 vs. HBOC=2.80±0.28, p<0.001). Conclusion A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.
    The Journal of Heart and Lung Transplantation 01/2015; 34(1):113-121. DOI:10.1016/j.healun.2014.09.021 · 5.61 Impact Factor
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    The Canadian journal of cardiology 10/2014; 30(10):S250-251. · 3.94 Impact Factor
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    The Canadian journal of cardiology 10/2014; 30(10):S251-252. · 3.94 Impact Factor
  • American Journal of Transplantation 06/2014; 14(S3):369. · 6.19 Impact Factor
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    The Journal of Heart and Lung Transplantation 04/2014; 33(4S):S109. · 5.61 Impact Factor
  • Transplantation 11/2013; 96(10S):S258-259. · 3.78 Impact Factor
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    ABSTRACT: Ex vivo heart perfusion (EVHP) has been proposed as a means to facilitate the resuscitation of donor hearts after cardiocirculatory death (DCD) and increase the donor pool. However, the current approach to clinical EVHP may exacerbate myocardial injury and impair function after transplant. Therefore, we sought to determine if a cardioprotective EVHP strategy that eliminates myocardial exposure to hypothermic hyperkalemia cardioplegia and minimizes cold ischemia could facilitate successful DCD heart transplantation. Anesthetized pigs sustained a hypoxic cardiac arrest and a 15-minute warm ischemic standoff period. Strategy 1 hearts (S1, n = 9) underwent initial reperfusion with a cold hyperkalemic cardioplegia, normothermic EVHP, and transplantation after a cold hyperkalemic cardioplegic arrest (current EVHP strategy). Strategy 2 hearts (S2, n = 8) underwent initial reperfusion with a tepid adenosine-lidocaine cardioplegia, normothermic EVHP, and transplantation with continuous myocardial perfusion (cardioprotective EVHP strategy). At completion of EVHP, S2 hearts exhibited less weight gain (9.7 ± 6.7 [S2] vs 21.2 ± 6.7 [S1] g/hour, p = 0.008) and less troponin-I release into the coronary sinus effluent (4.2 ± 1.3 [S2] vs 6.3 ± 1.5 [S1] ng/ml; p = 0.014). Mass spectrometry analysis of oxidized pleural in post-transplant myocardium revealed less oxidative stress in S2 hearts. At 30 minutes after wean from cardiopulmonary bypass, post-transplant systolic (pre-load recruitable stroke work: 33.5 ± 1.3 [S2] vs 19.7 ± 10.9 [S1], p = 0.043) and diastolic (isovolumic relaxation constant: 42.9 ± 6.7 [S2] vs 65.2 ± 21.1 [S1], p = 0.020) function were superior in S2 hearts. In this experimental model of DCD, an EVHP strategy using initial reperfusion with a tepid adenosine-lidocaine cardioplegia and continuous myocardial perfusion minimizes myocardial injury and improves short-term post-transplant function compared with the current EVHP strategy using cold hyperkalemic cardioplegia before organ procurement and transplantation.
    The Journal of heart and lung transplantation: the official publication of the International Society for Heart Transplantation 07/2013; 32(7):734-743. DOI:10.1016/j.healun.2013.04.016 · 5.61 Impact Factor
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    ABSTRACT: We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-l-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.
    Canadian Journal of Physiology and Pharmacology 01/2013; 91(1):45-55. DOI:10.1139/cjpp-2012-0215 · 1.55 Impact Factor
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    ABSTRACT: It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.
    Canadian Journal of Physiology and Pharmacology 07/2012; 90(8):995-1004. DOI:10.1139/y2012-034 · 1.55 Impact Factor
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    ABSTRACT: Low plasma high-density lipoprotein cholesterol (HDL-C) concentration is associated with the metabolic syndrome (MetS) and increased prevalence of cardiovascular disease (CVD). Animal and human studies report infusion of apolipoprotein A-1 (apoA-1) can reduce endothelial dysfunction, and/or induce regression of atherosclerosis. However, the direct mechanisms underlying the vascular benefits of either apoA-1 or HDL-C remain unclear. In this study, we assessed the ability of reconstituted HDL (rHDL) to improve vascular complications of MetS, including left ventricular (LV)-hypertrophy, arterial cholesterol deposition and myocardial lesion development. Obese insulin resistant (IR) JCR:LA-cp rats were infused with rHDL (0.4 mg/kg) over 3 days before assessing cardiac function (Echocardiography) at days 7 and 50 post-infusion, as well as haematoxylin and eosin staining of myocardial lesions at day 50. Acute ex vivo arterial cholesterol deposition was assessed with acute infusion of rHDL ex-vivo. Infusion of rHDL partially corrected abnormal diastolic compliance (18%; *p<0.05) and improved parameters of cardiac function in IR rats. Further, acute rHDL infusion in carotid vessels reduced remnant lipoprotein associated-cholesterol deposition (30-86%; **p<0.01) ex vivo in IR and male Wistar rats and reduced (41%; *p<0.05) the frequency of early-stage myocardial lesions in IR rats. Short-term infusion of rHDL may beneficially reduce chronic vascular sequelae of MetS, including temporary improvement in LV-dysfunction, acute reduction of acute arterial cholesterol deposition and the development of early-stage myocardial lesions in the JCR:LA-cp rat.
    Atherosclerosis 03/2012; 222(2):402-8. DOI:10.1016/j.atherosclerosis.2012.03.006 · 3.97 Impact Factor
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    ABSTRACT: Various procedures such as angioplasty, thrombolytic therapy, coronary bypass surgery, and cardiac transplantation are invariably associated with ischemia-reperfusion (I/R) injury. Impaired recovery of cardiac function due to I/R injury is considered to be a consequence of the occurrence of both oxidative stress and intracellular Ca(2+)-overload in the myocardium. These changes in the ischemic myocardium appear to activate both extracellular and intracellular proteases which are responsible for the cleavage of extracellular matrix and subcellular structures involved in the maintenance of cardiac function. It is thus intended to discuss the actions of I/R injury on several proteases, with a focus on calpain, matrix metalloproteinases, and cathepsins as well as their role in inducing alterations both inside and outside the cardiomyocytes. In addition, modifications of subcellular organelles such as myofibrils, sarcoplasmic reticulum and sarcolemma as well as extracellular matrix, and the potential regulatory effects of endogenous inhibitors on protease activities are identified. Both extracellular and intracellular proteolytic activities appear to be imperative in determining the true extent of I/R injury and their inhibition seems to be of critical importance for improving the recovery of cardiac function. Thus, both extracellular and intracellular proteases may serve as potential targets for the development of cardioprotective interventions for reducing damage to the heart and retarding the development of contractile dysfunction caused by I/R injury.
    International journal of cardiology 02/2012; 164(1). DOI:10.1016/j.ijcard.2012.01.103 · 6.18 Impact Factor
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    ABSTRACT: We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.
    Canadian Journal of Physiology and Pharmacology 02/2012; 90(2):249-60. DOI:10.1139/y11-128 · 1.55 Impact Factor
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    ABSTRACT: μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.
    Cell calcium 12/2011; 51(2):164-70. DOI:10.1016/j.ceca.2011.12.006 · 4.21 Impact Factor
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    ABSTRACT: Chemotactic movement of myofibroblasts is recognized as a common means for their sequestration to the site of tissue injury. Following myocardial infarction (MI), recruitment of cardiac myofibroblasts to the infarct scar is a critical step in wound healing. Contractile myofibroblasts express embryonic smooth muscle myosin, α-smooth muscle actin, as well as collagens I and III. We examined the effects of cardiotrophin-1 (CT-1) in the induction of primary rat ventricular myofibroblast motility. Changes in membrane potential (E(m)) and Ca(2+) entry were studied to reveal the mechanisms for induction of myofibroblast migration. CT-1-induced cardiac myofibroblast cell migration, which was attenuated through the inhibition of JAK2 (25 μM AG490), and myosin light chain kinase (20 μM ML-7). Inhibition of K(+) channels (1 mM tetraethylammonium or 100 μM 4-aminopyridine) and nonselective cation channels by 10 μM gadolinium (Gd(3+)) significantly reduced migration in the presence of CT-1. CT-1 treatment caused a significant increase in myosin light chain phosphorylation, which could be inhibited by incubation in Ca(2+)-free conditions or by application of AG490, ML-7, and W7 (100 μM; calmodulin inhibitor). Monitoring myofibroblast membrane potential with potentiometric fluorescent DiBAC(4)(3) dye revealed a biphasic response to CT-1 consisting of an initial depolarization followed by hyperpolarization. Increased intracellular Ca(2+), as assessed by fluo 3, occurred immediately after membrane depolarization and attenuated at the time of maximal hyperpolarization. CT-1 exerts chemotactic effects via multiple parallel signaling modalities in ventricular myofibroblasts, including changes in membrane potential, alterations in intracellular calcium, and activation of a number of intracellular signaling pathways. Further study is warranted to determine the precise role of K(+) currents in this process.
    AJP Heart and Circulatory Physiology 05/2011; 301(2):H514-22. DOI:10.1152/ajpheart.01041.2010 · 4.01 Impact Factor
  • Larry V. Hryshko
    Comprehensive Physiology, 12/2010; , ISBN: 9780470650714
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    ABSTRACT: Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.
    Journal of Biological Chemistry 10/2009; 285(4):2554-61. DOI:10.1074/jbc.M109.059162 · 4.60 Impact Factor
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    ABSTRACT: Na(+)/Ca(2+) exchangers (NCXs) promote the extrusion of intracellular Ca(2+) to terminate numerous Ca(2+)-mediated signaling processes. Ca(2+) interaction at two Ca(2+) binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca(2+) regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca(2+) interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca(2+) binding. The carboxylate residues that coordinate two Ca(2+) in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca(2+) regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca(2+) binding site to change Ca(2+) binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca(2+) regulatory behavior.
    Journal of Molecular Biology 02/2009; 387(1):104-12. DOI:10.1016/j.jmb.2009.01.045 · 3.96 Impact Factor
  • Biophysical Journal 02/2009; 96(3). DOI:10.1016/j.bpj.2008.12.1637 · 3.83 Impact Factor

Publication Stats

988 Citations
252.37 Total Impact Points

Institutions

  • 2012–2013
    • Hôpital St-Boniface Hospital
      Winnipeg, Manitoba, Canada
  • 1996–2013
    • University of Manitoba
      • • Department of Physiology
      • • Faculty of Medicine
      Winnipeg, Manitoba, Canada
  • 1999–2005
    • Simon Fraser University
      • Department of Molecular Biology and Biochemistry
      Burnaby, British Columbia, Canada
  • 1997–2004
    • St. Boniface Hospital Research
      • Institute of Cardiovascular Sciences
      Winnipeg, Manitoba, Canada
  • 1995–1996
    • University of California, Los Angeles
      • Department of Medicine
      Los Angeles, CA, United States
  • 1993
    • Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
      • Department of Medicine
      Torrance, California, United States