Myocardial Function Improved by Electromagnetic Field Induction of

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                                                                   OTR PROJECT CPCN                   EXHIBIT         B-21
ORIGINAL ARTICLI;
                                                                                                                                                          816



Myocardial Function Improved by
Electromagnetic Field Induction
of Stress Protein hsp70
ISAAC            GEORGE,'              MATTHEW          S. GEDDIS,2      ZACHARY    L1LL,'          HANA    L1N,3 TEODORO                GOMEZ?
MARTIN                 BLANK,4              MEHMET     C.   OZ,'    AND REBA   GOODMAN3     *
1 Department            of Surg~ry, Division of Cardiothoracic Surgery, Columbia University College of Physicians and Surgeons, New York,
New York
'Department  of Surgery, Division of Surgical Sciences, Columbia University College of Physicians and Surgeons, New York, New York
3 Department of Anatomy and Pathology, Columbia University College of Physicians and Surgeons, New York, New York
4Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York

Studies on myocardial function have shown thac hsp70. stimulated by an increase in temperature, leads co improved survival foHowing
ischemia-repetfusion (I-R). Low frequency electromagnetic ftelds (EMFs) also induce the stress protein hsp70, but without elevating
temperature. We have examined the hemodynamic changes in concert with EMF pre-conditioning and the induction of hsp70 to
determine whether improved myocardial function occurs following I-R injury in Sprague-Dawley rats. Animals were exposed to EMF
(60 Hz... !l- T! for 30 min prior to l-R. Ischemia was then induced by ligation of left amerior descending coronary artery (LAD) for 30 min.
         8
roHowed by 30 min of repetfusion. Blood and heart tissue levels for hsp70 were determined by Western blot and RNA transcription by
rtPCR. Significant upregulation of the HSP70 gene and increased hsp70 !evels were measured in response to EMFpre-exposures. Invasive
hemodynamics. as measured using a volume conductance catheter. demonstrated significant recovery of systolic comractiie function after
30 min of reperfusion following EMF exposure. Additionally, isovolemic relaxation, a measure of ventricular diastolic function, was
markedly improved in EMF-treated animals. In conclusion, non-invasive EMFinduction of hsp70 presel"Ved myocardial function and has the
potemia: to improve tolerance to ischemic injury.
   J. Cell. Physio!. 216: 816-823, 2008. \.~': 008 Wiley-Uss, Inc.
                                               2



Cardiovascular disease is the leading cause of morbidity and                    studies from Di Carlo et al. (1999) and Shallom et al. (2002)
mortality in the United States. accounting for 70-80% of deaths                 confirmed that cardiomyocytes were protected from anoxic
in men and women over the age or 65. Furthermore. congestive                    damage in EMF exposed chick embryos.
heart failure is the most common cause or hospitalization of the                   The induction of increased levels or hsp70 protein by low
elderly, and its incidence continues to increase (Schneider.                    rrequency EMF exposures offers multiple clinical advantages
 1999). In open and percutaneous revascularization procedures                   over thermal, chemical or gene-transfer methods of induction
(coronary artery bypass surgery and percutaneous coronary                       ror both patient and clinician. EMF stimulation of cytoprotecrive
interventions (PCls)) and in the treatment of myocardial                        proteins is a non-invasive procedure easily administered to the
infarction and heart failure, it is essential to protect                        patient. EMF-induced hsp70 does not turn off baseline protein
cardiomyocytes from the effects of hypoxia and ischemia (Bolli                  synthesis, in contrast to elevated temperature (Goodman et al..
et aI., 2004). Currently, myocardial protection can be                           1989). A significant increase in hsp70 stress protein is induced
accomplished by induction or the stress protein hsp70 through                   within 5 min at 14 orders of magnitude lower energy input than
the use or elevated temperature      (heat shock) (Currie et al..               thermal stress. Additionally, unlike thermal stress, the induced
 1993; Udelsman et aI., 1993; Nitta et al.. 1994; Plumier and                   protection can be restimulated even after the stress is already
Currie. 1996). Induction of stress proteins by heat to prevent                  present, and restimulation with even higher hsp70 levels can be
stroke and myocardial infarction during reperfusion has been                    induced by a different field strength. higher (800 mG) or lower
shown to partially protect the myocardium under ischemic                        (8 mG; Blank et al.. 1994; Lin et aI., 1997).
stress in a variety or models (Heads et aI., 1995; Mestril et aI.,
 1996; Plumier and Currie, 1996; Benjamin and McMillan, 1998;
Chong et aI., 1998; Cornelussen et aI., 1998). The use of heat
stress pre-treatment     leads to moderate increases in hsp70
levels. but does not improve ischemia tolerance in isolated                        Abbreviatidns: LVSP,left ventricular systolic pressure; LVdP/dt",.x.
hearts (Cornelussen et aI., 1998). Moreover. heat stress                           'eft ventricular; P-A Max Power. pre-load adjusted maximum
pre-treatment     (hyperthermia) is or limited clinical utility since              power; G, Gauss; rtPCR, reverse-transcriprase polymerase chain
it requires a temperature elevation to 42°C, a level impractical                   reaction.Contract grant sponsor: Robert I. Goodman Fund.
for clinical use or to achieve sufficient hsp70 increases.                         Contract grant sponsor: National Instiwte of Health;
    We have shown previously that 60 Hz electromagnetic fields                     Contract grant number: T32-HL07854.
(EMFs) upregulate the heat shock gene, HSP70 and induce                            *Correspondence to: Reba Goodman, Department of Pathology,
elevated levels of hsp70 protein in the absence of elevated                        Columbia University College of Physiciansand Surgeons, PH, Rm.
temperature (Goodman etal.. 1994; Goodman and Blank, 1998;                         15-90,630 W. I 68th St., New York, NY 10032.
Han et al.. 1998; Lin et aI., 1998, 1999, 200 I; Carmody et aI.,                   E-mail:rmg5@columbiaedu
2000). Of particular relevance, we previously elevated hsp70                       Received 5 February 2008; Accepted 4 March 2008
levels in cultured rodent cardiomyocytes using EMF                                 DOl: 10.1002/jcp.2146I
pre-treatment     (Goodman and Blank, 2002). Additionally.


.:.-::'   c F.   '/.     -   v   . S   S.    • ~   c
                                  MYOCARDIAL                   PROTECTION                       AND           EMF
                                                                                                                                                      817


    Extensive reports in the literature have shown that elevated
levels of hsp70 improve cardiac function after hypoxic stress
and ischemia-reperfusion    (I-R; Suzuki et aI., 1997, 2000, 2002).
In this set of experiments, we evaluated whether pre-treatment
                                                                          A                          Helmholtz Coils
with EMF-induced levels of hsp70 can preserve myocardial                               __           ---241.'''cm-
function after ischemia-reperfusion.

Methods
Animal care                                                               ~'cm {
                                                                          between
Male Sprague-Dawley rats (250-400 g) were selected as the                 levels
experimental species. To assure their health, the animals were
examined by the animal facility veterinarian upon arrival. Animals
were allowed to acclimate for at least 2 days and adjust to being
handled before randomization into the study. Animals were housed
in cages in an environmentally controlled room within the animal
care facility at Columbia University. Care and management of
rats was conducted according to facility standard operating
procedures. At the conclusion of the acclimation period animals
judged to be suitable for testing were assigned sequentially to
either treatment or control. This investigation conforms with the
Guide for the Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23, revised
                                                                                        r
                                                                                    114 em   thickness   .J
 1996). This study was approved by the Institutional Animal Care
and Use Committee of Columbia University.
                                                                           8
Electromagnetic    field exposures                                                                             ,~~               ~
All EMF exposures were performed prior to induction of ischemia.
The exposures described here are pre-treatment exposures. In
previous studies, we tested a variety of field strengths and
frequencies (Goodman etal., 1989; Wei etal., I990;Jin etal., 1997),
and eventually determined that a 60 Hz frequency and a fteld                                                                         --r
                                                                                                                                     ' ' CIll tniCk
strength of 8 µ.T consistently induced the highest level of
transcriptional activation of the HSP70 gene and the highest hsp70                                                         1
protein levels (reviewed in Goodman and Blank. 2002). In the                                                               I
studies reported here. animals were exposed to 60 Hz/a µ.T EMFs                                                            i
                                                                                                                         fr'cm
in a plastic exposure cage (16 cm x 24 cm) surrounded by
Helmholtz coils (19 gauge copper wire, 164 turns, 1.5 inches thick
                                                                                                                           i
                                                                                                                           I
                                                                                                                           I
covered with electrical tape). The system was designed and                                                                 !
calibrated (R. Cangialosi, Electro-Biology, Inc., Fairfield, NJ) to our
specifications for comfortably holding a large rodent; the cage
holding the rats was suspended in a plastic enclosure (Fig. I).
EMF conditions were set using a function generator (BK Precision
40 II A 5 MHz, Yorba Linda, CAl and digital multi meter                   Fig. I. EMFexposure system. Animals were exposed to 60 Hz/a µT
 (BK Precision 2706A). Rectal temperature was continuously                EMFsby Helmholtz coils (19 G copper wire, 164 turns, 1.5 inches thick
                                                                          covered with electrical tape; part A) that was contained within a
 monitored with a thermocouple probe (±O.I cC resolution;                 plastic exposure cage (part B). The EMFfield was perpendicular to the
 PhysiTemp., Cliffside Park. NJ). The digital multimeter was used to      exposure device.
 measure the field intensity and verify the systems operation. Field
 parameters were monitored with a Hitachi V-I 065 100 MHz
 oscilloscope and a calibrated inductive search coil. Exposure
                                                                          (pre-exposure) and after EMF exposure every 30 min up to
 conditions were monitored with a Sypris triaxial magnetic field
                                                                           120 min. Serum was spun down at 1,200 rpm for 10 min; packed
 meter (Model 4080, Bell Laboratories, Orlando, FL). Experiments
                                                                          red blood cells (RBCs) were flash-frozen for hsp70 protein
 were carried out at room temperature (approx. 25'C).
                                                                          analyses. and stored at -80'C.
Anesthesia
                                                                          Terminal tissue for HSP70 RNA determination
Rats were anesthetized with 2% isoflurane and mechanically
ventilated via a tracheostomy (Harvard rodent ventilator, model           Heart tissue (left ventricle; LV) was flash-frozen for analysis
683, South Natick, MA) throughout the duration of the                     of HSP70 RNA by reverse-transcriptase       polymerase chain
experiment.                                                               reaction (rtPCR). GAPDH confirmed loading concentrations.

Surgical Procedures                                                       Ischemia-reperfusion                protocol



                                                                                                                                                      j
Blood collection for hsp70 determination
                                                                          The hemodynamic effectS of EMF exposure on myocardial
The left femoral vein was cannulated with polyethylene tubing             function after I-R (EMF n = 10. Control n = 10) were measured
(00 0.965 mm, Becton Dickinson, Franklin Lakes, NJ) for                   using the following protocol. Pre-anesthetized   animals were
repeated blood draws. Rats were then randomized to EMF                    randomly assigned to EMF pre-treatment      and underwent EMF
exposure (60 Hz 8 µ.T) for 30 min (n = 6) or Control                      exposure (60 Hz, 8 µ.T) for 30 min. During this time period,
(no EMF exposure, n = 6). Blood was collected at baseline                 each individual animal was allowed to rest comfortably in the


JOURNAL OF CellULAR    PHYSIOLOGY
818                                                           GEORGE        ET AL.


      exposure device under quiet conditions. No medications were              Quantification   of bands on films from Western blots and
      administered prior or during this time. Animals designated as         rcPCR. The films from Western         blots and rtPCR analyses
      controls received no exposure and their cages were maintained         were scanned into a computer. The density of the bands was
      in an EMF-free area and monitored with a Sypris triaxial              measured using image analysis software (ImageJ v 1.38, NIH).
      gauss meter. At the conclusion of 30 min, all rats (both exposed         Statistical analysis. Continuous variables are expressed
      and control) were anesthetized with 2% isoflurane and                 as mean ± standard error and compared using two-tailed
      mechanically ventilated on 2% isoflurane after a tracheostomy.        independent t-testing with Levene's Test for Equality or
      A 2 Fr volume conductance catheter (Millar Instruments,               variances. Categorical variables were compared by      l   tests.
      Houston, TX) was inserted into the left ventricle (LV) for            Paired t-testing was used to evaluate significance within groups
      continuous pressure-volume     (PV) tracings immediately              at multiple time points. For all analyses, a P-value of less than
      following right carotid artery cannulation. After left                0.05 was considered statistically significant. All analysis was
      thoracotomy, coronary ischemia was created by ligating the            performed using SPSS software (v. 11.5, Chicago, IL).
      left anterior descending (LAD) coronary artery with a
      5-0 polypropylene suture for a total of 30 min before release of      Results
      the suture. Reperfusion was monitored foran additional 30 min.        EMF-induced     hsp70 levels
      Hemodynamics (Chart for Windows v5, ADI Instruments,
      Colorado Spring, CO) were recorded at baseline                        As described above, rats were exposed to EMF (60 Hz, 8 µ.T)
      (pre-ischemia), 30 min of ischemia, and at I, 10, 20, and 30 min      for 30 min, blood samples were collected from the left femoral
      after reperfusion. Upon completion of the experiment, the             vein from both EMF-pre-treated and unexposed (control) rats
      heart was excised, and the LV was sectioned for histology.            just before (baseline) and immediately after EMF exposure
      Ischemic and non-ischemic portions of the LVwere flash-frozen         (time 0) and at 30, 60, 90, and 120 min. Blood samples were
      for extraction of protein for Western blot analysis of hsp70          prepared for protein extraction and subsequent Western blot
      levels and RNA extraction for reverse-transcriptase                   analyses of hsp70. As seen in Figure 2A, the levels of hsp70
      polymerase chain reaction (rtPCR) to determine upregulation           protein were significantly elevated following 30 min of EMF
      of the HSP70 gene.                                                    pre-exposure, and the increase in protein level was sustained
                                                                            for 120 min post-exposure     (P < 0.05 vs. Control, n = 6 per
                                                                            group. Fig. 2A); peak levels were reached at 30 min. Low
      Hemodynamics       and pressure-volume       analysis                 baseline (constitutive) levels of hsp70 protein exist under
      Hemodynamic determinations were made on all rats                      non-stressed conditions. After EMF exposure, these levels
      undergoing I-R (n = 20). LV end-systolic pressure (LVSP), LV          were significantly elevated (>40%).
      end-diastolic pressure (LVEDP), and LVvolume were measured
                                                                            EMF-induced     transcript     levels for HSP70 RNA
      using the Millar conductance catheter placed into the LV across
      the aortic valve. End-systolic volume (ESV) and end-diastolic         Terminal myocardial tissue was collected for determination of
      volume (EDV) were measured using standard techniques (Ito             HSP70 RNA by rtPCR. Transcript levels of HSP70 RNA were
      and Bassett, 1983). Cardiac output (CO), arterial elastance (Ea),     significantly increased in terminal myocardial tissue extracts
      and pre-load adjusted maximum power (P-A Max Power) were              in response t:o EMF pre-exposure    as compared to controls
      comput:ed using a pressure-volume       analysis program (PVAN v.     (P < 0.05 vs. Control, n = 6 per group), demonstrating
      3.2, Millar Instruments). The time constant of LV isovolumetric       upregulation of the HSP70 gene (Fig. 2B). There was no increase
      pressure relaxation, 7, was calculated using the logarithmic          in HSP70 transcript levels in Control animals during the same
      method described by Raff and Glantz (1981). In experiments            time period. These results confirm previous literature that EMF
      where animals had undergone                                           induces upregulation of the HSP70 gene and increases hsp70
      ischemia-reperfusion,     the hemodynamic and pressure-volume         protein levels (Un et aI., 1997, 1998, 1999, 200 I).
      analyses indicated no adverse events or mortality associated
      with EMF exposure.                                                    Ischemia-reperfusion:        hsp70 levels
          Protein sample preparation.       Protein was extracted from
                                                                            Levels of hsp70 were increased in both non-ischemic (N) and
      myocardial tissue and packed RBCs using methods previously
                                                                            ischemic (I) left ventricle (LV) tissue after the termination of
      described (Lin et aI., 1998; Carmody et aI., 2000). Protein
                                                                            I-R in EMF-exposed samples as compared to Control animals
      concentrations were determined by Bradford assay (Bio-Rad
                                                                            (P < 0.05 vs. Control, n = lOpeI' group; Fig. 3A). rtPCR analysis
      Laboratories, Hercules, CA).
                                                                            of terminal LV tissue from ischemic (I) and non-ischemic (N),
          Western blot. Equivalent (30 µ.g) amounts of protein were
      separated by gel electrophoresis      on 10% polyacrylamide gels      showed increased RNA transcript levels in EMF pre-exposed
                                                                            tissue as compared to controls (P = 0.055 vs. Control, n = 10
       using appropriate molecular weight markers and transferred to
                                                                            per group; Fig. 3B). The current results confirm that, in this
       PVDF membrane for immunoblotting. Blots were probed
                                                                            ischemia-reperfusion     model, EMF exposure upregulates the
      with anti-hsp70 antibody (I: I0,000; kindly provided by
                                                                            HSP70 gene and significantly increases hsp70 levels.
       Dr. Richard Morimoto, Northwestern          University). The blots
      were then stripped and reprobed with anti-f3 actin (I: 1,000,         Ischemia-reperfusion:        Hemodynamics
       Sigma-Aldrich, St. Louis, MO) to confirm equivalent loading.
       Visualization was by the ECL detection system as previously          No adverse events or mortality were associated with EMF
       described (Lin et aI., 1998).                                        exposure. The mean temperatures        before and after 30 min
          Reverse-transcriptase        polymerase       chain reaction      of EMF exposure were 36.1 ± 0.2nC and 36.0 ± 0.2"C,
       (rcPCR). Total RNA was extracted from non-ischemic and               respectively, versus 36.0 ± 1.1cC in Control rats (EMF vs.
       ischemic left ventricular (LV) heart tissue using Trizol reagent     Control, P = NS). The hemodynamics at baseline, after 30 min
       (Life Technologies, Inc., Rockville, MD). Total RNA (0.1 µ.g)was     or coronary ischemia and after 10-30 min of reperfusion, are
       processed directly to cDNA synthesis using the TaqMan"               summarized in Table I. LVSP was significantly improved
       Reverse Transcriptase Reagents kit (Applied Biosystems,              beginning at 10 min of reperfusion until 30 min in EMF-treated
       Foster City, CA). All PCR primers and TaqMan probes were             animals versus Control (Fig. 4A). No significant differences
      designed using PrimerExpress software (Applied Biosystems)            were seen in LVEDP, ESV, and EDV. Coronary ischemia
      and published sequence data from the NCBI database (Un et al..        produced a significant reduction in LV dP/dc",.x (a measure of
      2001).                                                                ventricular contractility) after 30 min. Recovery of LV dP/dc",ax


      JOURNAL OF CELLULAR PHYSIOLOGY
                                          MYOCARDIAL                                                    PROTECTION                       AND                        EMF
                                                                                                                                                                                                                                               819




 A                                      Minutes                                                                  A                    Control                                                   EMF
          BL 0        30 60 90 120                BL         o     30 60 90 120                                                  N       I                N         I          N        I       N       I          N
 92Kd--F ".
 53Kd__
           . "~
             --                   -
                                                                                                        hsp70     K
                                                                                                                 92 d--Ilf-=".';B
                                                                                                                 53Kd __         '" ..... ~.               '., ....•                                          .:.;.c .... ~
                                                                                                                                                                                                                                    hsp70

 ;;~=:~.. ---.l I
                      Control                                       EMF
                                                                                                   I                  ~-"-I s...--..
                                                                                                                                   I                                                                                                (i-actin

                                                                                                                            j(i;"'
                                                                                                                                     24000           0
                                                                                                                                                     •
                                                                                                                                                            Conlml (11=10)
                                                                                                                                                            EMF(.=10)
                                                                                                                                                                                                     • P < 0.05 vs Control


                  o     Control (.=6)                                      '\I < o.os   lIS   Control                  In    =2      21 (XXl                                   *
                                                                                                                       -::I                                                    ~
                  •     EMF(n=5)
                                                                                    *                                      ~ eo      18lXXl1                                   i                            T                 T
                                                                                                                       - ..
                                                                                T                                      _<':I,                                             -
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                                                                                                                                      6000

                                                                                                                             S        3000
                      Bl     0             30                60                90              120
                                                                                                                                             0
                      L..--J
                      E10IFexpcsure
                                                                                                                                                                   Ischemic                           Non-Ischemic
                          30 min
                                        Exposure Time (min)

                                                    61'\1 < o.os vs Control              *
                                                                                                                           B                                        '0
                                                                                                                                                         u..u...J::u..=
                                                                                                                                                         ====§==§
                                                                                                                                                                                   '0            ..
                                                                                                                                                                                            u...u...-
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 B                                                                                                                                                       ww(.)w(.)                          w       w (.) w                    0
                                                                                                                                                                                                                              (.)
                                                                                                                                                                                            A                                       HSP70
                                         ~~ 51
                                         >;:::
                                         G:lc
                                         - '"
                                         <t ...     4                                                                                                n:X:X:t:-J'                                    iii                       i i GAPDH
                                         z...
                                         c: ~  J1                                                                                                              Ischemic                         Non-Ischemic
                                         ~;:::   I
                                         ~i
                                         ::c~
                                                    2j
                                                         j                                                                            1400 )         0        Conlrol (n:10)                         • p    = 0.055 VS Control
                                                                                                                                                     •        EMF (n=10)       *
                                                    Ii
                                                         i I               I                                                          1200       J
                                                                 Control                EMF                                           1000       J
                                                                 (n::6)                 (n=6)
                                                                                                                                                                   T
Fig. 2. hsp70 protein levels and HSP70 RNA transcript levels are
progressively elevated after a 3D-min EMFexposure. A: In response to
30 min of EMF exposure (60 Hz/8 .... hsp70 levels in blood were
                                       T).
                                                                                                                                        :j
                                                                                                                                         i
                                                                                                                                        200
                                                                                                                                                               r
                                                                                                                                                               I
                                                                                                                                                                        -
significantly elevated at 30 min and sustained for 120min as compared
to unexposed control samples (*P < 0.05 vs. Control). B: HSP70 RNA
transcript levels were significantly increased in terminal myocardial                                                                                              Ischemic                                 Non-Ischemic
tissue extracts in response to EMF exposure as compared to
unexposed control samples (*P < 0.05 vs. Control).
                                                                                                                Fig. 3. Ischemia-reperiusion: hsp70 protein levels and HSP70 RNA
                                                                                                                transcript levels were increased significantly following EMFexposure.
                                                                                                                A: Levels of hsp70 were increased in both non-ischemic (N) and
                                                                                                                ischemic (I) blood samples after ischemia-reperiusion. However,
 was only seen in EMF-treated groups compared to Control                                                        the hsp70 increase in ischemia samples was significant as compared
 from 10 until 30 min after reperfusion (Fig. 4B). Pre-load                                                     with non-ischemic and control samples (*P<0.05 vS. Control).
 adjusted maximum power, a load and heart rate independent                                                      B: A significant increase in HSP70 transcript levels was seen in the
 index of systolic contractile function (the ability of the heart to                                            EMF pre-exposed terminal myocardium tissue from ischemia (I) left
                                                                                                                ventricular tissue as compared with non-ischemic and control
 eject blood), was similarly improved in EMF-treated rats after                                                 samples (*p= 0.055 vs. Control).
 reperfusion, as shown in Figure 4C. These results, interpreted
 collectively, show that overall organ perfusion was increased
 during reperfusion (manifested by higher LVSP). The ability to
 increase ventricular pressure at the time of reperfusion despite                                                ventricular wall compliance, allowing faster and greater
 ischemic injury, without changes in blood volume, ventricular                                                   diastolic filling. Greater diastolic filling and relaxation of the
 dimensions, or exogenous pharmacologic agents, implicates a                                                     ventricular wall improves cardiac output, lessens myocardial
 direct mechanism to improve cardiac myocytes, and myocardial                                                    oxygen consumption, and increases the overall efficiency of the
 systolic contractility. In addition, the lack of ventricular                                                    heart. Although no significant changes in arterial elastance were
 dilatation and absence of end-diastolic pressure elevation                                                      seen, a strong recovery trend of cardiac output to 71 % of
 implies normal end-systolic and end-diastolic pressure volume                                                   baseline with EMF versus only 43% in Control was observed.
 relationships, in contrast to acutely failing hearts, which                                                     Adverse changes in arterial elastance would potentially affect
 undergo pressure-volume       overload, enlargement of ventricular                                              long-term remodeling of central arteries, leading to multi-organ
 dimensions, and elevated end-diastolic pressures.                                                               dysfunction (most notably, renal failure).
    The contribution of diastolic function was also evaluated by                                                    In summary. a clear effect on systolic contractile function in
calculating the time constant of LV isovo/umetric relaxation. A                                                 EMF-treated animals was found after reperfusion, as shown by
slight, but statistically significant decrease in 'T (a measure of                                              increases in global indices of contractile function (LV dP/dtmax
diastolic function) was observed throughout the course of                                                       and pre-load adjusted maximum power). These changes
reperfusion after EMF treatment (Fig. 5), indicating improved                                                   occurred without evidence of concurrent LV hypertrophy or at

JOURNAL OF C:FLLULAR PHYSIOLOGY
820                                                                                      GEORGE                       ET AL.


      TABLE      I. Hemodynamicsand pressure-volume analysis

                                          Pre-isch              30 minischemia                 I minreperfusion              10 min reperfusion    20 min reperfusion     30 min reperfusion

      Heart rate (bpm)
             Control                     275 ± 7                    249± III                        220± 10'                      221 ±9'              224±8'                   226±8'
               EMF                       269 ± 10                   260± 9                          246±8'                        259 ± 10'            254± II~                 244± 10
      LVEDP (mmHg)
              Control                    9.9± 1.6                   15.7±3.11                       13.3±2.1                      13.2±2.0             13.1± 1.9                13.2± 1.8
               EMF                       9.7 ± 1.2                  15.1±2.l1                       13.7±2.1                     15.28± 2.1            16.4±2.9                 16.3±2.9
      Cardiacoutput (ml/sec)
            Control                      667± 210                   385 ± 109                       214±42                        286 ±95              279± 100                 375± 136
               EMF                       467± 100                   318±60                          224±28                        334±57               328±50                   297±47
      E. (mmHglml)
              Control                    106±23                      148±51                         154±40                        151±42               177±45                   164±45
               EMF                       105± 23                     105±29                         108±9                         112± 15              109± 17                  115± 17

      EMF. elecuoma.gnetic   field; LVEDP, left   ventricular end..diastolic   pressure; 1;. arterial   elastance .
      •p < 0.05 vs. Control.
      'p < 0.01 vs. Control.
      ip < 0.05 vs. Baseline.
       • p < 0.05 vs. 30 min Ischemia.


      the expense of reduced diastolic function, and without                                                          exposure, suggesting that diastolic function was not adversely
      increased ventricular dimensions, which would have been                                                         affected in order to maintain hemodynamics.
      expected with pressure-volume     overload seen after ischemic                                                     The changes in ventricular function appear to relate primarily
      injury. The changes in diastolic function may also reflect an                                                   to the ability of the myocyte to tolerate ischemia and prevent
      effect of hsp70 on stabilization of cellular structure.                                                         injury after reperfusion. After reperfusion, in addition to
                                                                                                                      molecular mechanisms that permanently and directly cause cell
      Discussion                                                                                                      death, cardiomyocytes face secondary insults in the form of
                                                                                                                      inadequate coronary perfusion as a result of impaired
      The cytoprotective     effect of stress response proteins,                                                      hemodynamics (Le., lower systolic pressures, decreased
      specifically hsp70, as a cellular defense mechanism, has been                                                   cardiac output, reduced contractility). The degree of ischemic
      well described in the literature (Westerheide        and Morimoto,                                              tolerance displayed by EMF-exposed animals to resist the usual
      2005; Uu et aI., 2007; Zhao et aI., 2007; Brocchieri et aI., 2008).                                             hemodynamic derangement after reperfusion is a likely factor in
      Myocardial preservation after HSP70 gene transfection was                                                       both cell survival and augmentation of existing cell functional
      first reported by Suzuki et al. (1997) who showed that increased                                                units. Subsequently, reduction of infarction, salvage of
      hsp70 protein resulted in improved recovery of coronary flow,                                                   underperfused myocytes at risk for necrosis, and increased
      maximum LV dP/dt, and LV developed pressures. In isolated rat                                                   myocardial perfusion are all expected cellular changes (Heads
      heartS undergoing 30 min of ischemia and 30 min of reperfusion,                                                 etal .. 1995; Mestril et:al .. 1996; Plumier and Currie, 1996; Suzuki
      post-ischemic mitochondrial respiratory control indices                                                         et aI., 1997; Benjamin and McMillan, 1998; Chong et aI., 1998;
      (NAD' - and FAD-linked respiration) were improved in                                                            Cornelussen et aI., 1998; Jayakumar et aI., 2000, 200 I; Okubo
      HSP70-transfected animals Oayakumar et aI., 200 I). In a similar                                                et aI., 200 I). Improvements in systolic function can serve to
      study, HSP70 gene transfection attenuated creatinine kinase                                                     interrupt the cycle of death which normally follows impaired
      release and preserved coronary endothelial response to                                                          contractility and prolonged hypoperfusion. Finally, subtle
      vasodilatory agents Oayakumar et al.. 2000). Finally, infarct                                                   changes in ventricular compliance (Le., a reduction in -:) may be
      reduction of almost 50% was demonstrated           in HSP70-                                                    critical for diastolic filling after reperfusion, especially in the
      transfected rabbit hearts after ischemia-reperfusion        (Okubo                                              setting of ischemia and increased myocardial oxygen
      et aI., 200 I).                                                                                                  consumption/wall stress.
          In the experiments reported here, we have applied 60 Hz,
      8 µ.T EMFs to elevate hsp70 levels. Based on prior studies, we
                                                                                                                      Possible   mechanisms       for improved     myocardial
      hypothesized that EMF exposures would increase levels of
                                                                                                                      function   by EMF
      hsp70 and be cytoprotective       after ischemia-repetfusion.
      Following ischemia-reperfusion       after EMF exposure, we found                                               Recent studies have shown strong correlation between
      significantly increased expression of the HSP70 gene and                                                        myocardial calcium handling to the function of hsp70-for
      elevated hsp70 protein levels, and myocardial function (both                                                    example, deleting the inducible 70 kDa heat shock genes impairs
      systolic and diastolic) was significantly improved.                                                             cardiac contractile function and calcium handling associated
                                                                                                                      with hypertrophy (Okubo etal .. 200 I; Kim eta!.. 2006). The Nal
                                                                                                                      Cah exchanger, known to play an important determinant of
      EMF-exposure     improves contractile                             function         and
                                                                                                                      myocardial contractility in heart failure, is acted on by hsp70,
      reduces reperlusion    injury
                                                                                                                      leading to desensitization by a reduction in Vmax (Kiang et aI.,
      The hemodynamic changes due to EMF exposure prior to                                                             1998). Hsp70 may also alter protein kinase A. protein kinase C,
      ischemia are slight. However, our hemodynamic findings after                                                    and phospholipase A2 (Ding et aI., 1998); it is conceivable that
      ischemia-reperfusion     demonstrate that EMF-induction of                                                      hsp70 also modulates other components of calcium-handling,
      elevated hsp70 levels significantly reduced cardiac injury.                                                     such as SERCA2a and phosphorylation        of the ryanodine
      Systolic contractile function, as measured by LV dP/dt",ax and                                                  receptor (Marx et aI., 2000). These results suggest interaction
      left ventricular systolic pressure (LVSP), was significantly                                                    of hsp70 with elements of the calcium release mechanism that
      increased in exposed animals compared to control animals,                                                       result in enhanced myocardial contractility.
      despite reductions in all groups after 30 min of ischemia.                                                         Induction of hsp70 levels by rapid elevation of temperature
      Pre-load adjusted power, a pre-load and rate-independent                                                        also improves Cytoskeletal-based cell survival pathways 0Nei
      measure of systolic function, was also improved throughout                                                      et aI., 2006). Maintenance of cell architecture associated with
      the reperfusion period. Although LVEDP did not change,                                                          hsp70 has been reported in the literature previously 0Nei et aI.,
      isovolemic relaxation was substantially improved after EMF                                                      2006), and anti-apoptotic mechanisms of stress response


      JOURNAL OF CELLULAR PHYSIOLOGY
                                                   MYOCARDIAL                                   PROTECTION                         AND           EMF                               821




         140        -
 A       130        -
                        -0- - Ccntrol (n:l0)
                                 EMF (n:1DI
                                                                                                                  43
                                                                                                                       1   --0--   Cllnlrol (n=IO)
                                                                                                                  40"      -       EMF (n=10)        t
         120                                                                                                      'rl
                                                                                                                                                      -, + -----t-----f -
                                                                                                                                                      ~
                                                                                                                                                         r
                                                                                                                                                       ,<1>----  .*   -   *
         110
         100
                                                                   '/~Jr---~
                                                                                                                  34
                                                                                                                                                 ~

                                                               t
                                                                                                                  31
          90                                            __ -            _             J                   Cii
                                                                                                          §..     28
                                                                                                                                           ~,
                                                                                                                                     ,
          80
                                                                                                           ....   25
          70                                                                                                      22       ~~
          60
                                                                                                                  19
          50 l-Ischemia-                            _      Repertusion-
                                                                                                                  16
          40'                                                                                                              -Ischemia--                 -Reperfusion-
              o     10                 20          30          40      50            60              70           13            "                          "
                                                                                                                               10  20                30   40   50  60         70
                                                   Time(min)
 8                                                                                                                                                   Time (min)
       , lIOOO  1 -     -0- -    Control (11=10)
~        9000 1     -            EMF (n=10)
                                                                                                          Fig. S. Ischemia-reperfusion: diastolic function was significantly
 ~      8ooo~                                                                                             improved in response to EMF exposure. Diastolic function, or
 :::                                                           *                                          isovolumetric ventricular relaxation as measured by Tau (T), was
                1
                '



 §      7000                                                                                              significantly improved throughout Reperfusion after 30 min
-;;     6000'                                                                                             of EMF exposure ep<O.OS vs. Baseline, ·P<O.OS vs. Control).

:j      50001
                                                        --~} ---·i ---1
!5      4000    1
~~:j                                                                                                      phosphorylation and activation of the upstream kinase SEK,
                                                                                                          or by inhibition of stress-induced suppression of jNK
                                                                                                          dephosphorylation     (Meriin et aI., 1999; Yaglom etal.. 1999; Park
                I
        1000 +--Ischemia-                           -Repertusion-                                         et aI., 200 I). A secondary effect of HSP70 gene upregulation is
           0'                                       ,                                  ,                  increased manganese superoxide dismutase activity, which
             o    10    20                         30          40      50             60             70   serves to limit mitochondrial apoptosis in ischemia-reperfusion
                                                   Time(min)                                              (Suzuki et al.. 2002). Interestingly. MDA and LDH levels,
 c                                                                              o Centrol   (n=1 O)
                                                                                                          markers of cardiac injury. were unchanged in our experiments
                                                                                                          (data not shown): this suggests that EMF does not diminish the
                                                                                                          extent of cardiac injury but rather enhances the contractile
                          T
                          !
                                                                                •   EMF!n=10)
                                                                                                          function of remaining myocytes.
                                                                            *
                             i
                             I
                                                               *                                 *
                                                                                                          EMF and biological interactions
                                                                                                          EMF interaction with cells and tissues has been extensively
                                                                                                          studied in vivo and in vitro (reviewed in Goodman and Blank.
                                                                                                          2002; Blank and Goodman, 2004. 2007). EMF is known to
                                                                                                          induce elevated DNA transcript levels of several genes
                                                                                                          including HSP70 as well as elevated levels ofhsp70 protein in the
                                                                                                          absence of increased temperature      (reviewed in Goodman an'd
                                                                                                          Blank. 1998). The interaction mechanism of EMF with DNA in
                        30                  1            10            20                   30            cells and tissues to stimulate protein synthesis remains
               -Ischemia.... •                             Reperfusion                                •   unknown. Several theoretical approaches to EMF mechanism
                                                   Time (min)                                             have been proposed including cyclotron resonance (Liboff,
                                                                                                           1985; Lednev, 1991) and forced vibration ofjons (Panagopoulos
                                                                                                          et aI., 2002). There is evidence from biochemical reactions that
Fig. 4. Ischemia-reperfusion: Contractile function was significantly                                      EMFs can accelerate electron transfer and move within DNA
improved in response to EMF exposure. Contractile (systolic)                                              (Blank and Soo, 200 I: Goodman and Blank, 2002; Blank and
function was significantly increased after ischemia-reperfusion in
response to 30 min of EMF exposure, as measured by ventricular                                            Goodman, 2004, 2007).
systolic pressure (LVSP; A), left ventricular dP/dt (LV dP/dt",u; B),                                        An important clue to EMF stimulation of biosynthesis comes
and pre-load adjusted maximum power (P-A Max Power; C;                                                    from identification of a specific EMF-sensitive DNA sequence
tp<O.OS v•. Baseline, ip<O.OS vs. Ischemia 30, ·P<O.OS vs. Control,
                                                                                                          on both the c-myc and the HSP70 gene promoters (Lin et aI.,
lp<O.OI vs. Control, ~P<O.005 vs. Control).
                                                                                                           1999, 200 I). The HSP70 promoter has three nCTCT n
                                                                                                          recognition motifs/response elements (- 158 to - 203 relative
                                                                                                          to the transcription initiation site) that are EMF-sensitive (Un
proteins, such as attenuation of the ASK-jNKfp38 signaling                                                et aI., 1999). The heat shock element (HSE), lying between
cascades (Fan et aI., 2005), has beeh documented. There is also                                            - 180 and - 203, is required for induction of HSP70 gene
evidence that hsp70 has a direct effect on apoptosis by                                                   expression by EMFs (Lin et aI., 1999, 200 I). Mutation of
preventing caspase-3 activation, PARP cleavage, DNA                                                       the nCTCTn sequences (EMRE, electromagnetic          response
laddering, and cell death in vitro, but its actions appear to be                                          elements) eliminates the EMF sensitivity of the HSP70
downstream of cytochrome C release (Westerheide and                                                       promoter (Lin et aI., 200 I). The EMF domain and the heat shock
Morimoto, 2005). The ATP-binding domain of HSP70                                                          domain function independently. This is an HSF-I dependent
suppresses SAPKfjNK activation. either through suppression of                                             process (Lin et aI., 1998, 1999: Un et al.. 200 I). Furthermore,


JOURNAL     OF CELLULAR PHYSIOLOGY
822                                                             GEORGE          ET AL.


      the HSE in the heat shock domain is not interchangeable with              or following surgery. The exposure technology is entirely non-
      the HSE in the EMF domain (Lin et aI., 200 I). nCTCT n                    invasive. This device is portable, weighs about one pound. and
      sequences, placed upstream of CAT or luciferase reporter                  can be easily applied by any technician.
      constructs (that were otherwise unresponsive to EMFs) were                   The use ofEMFs for the induction ofhsp70 for post-ischemia-
      transfected into HeLa cells and exposed to EMFs. Protein                  reperfusion treatment has clear advantages over the invasive
      extracts from EMF-exposed transfectants had significant                   elevated temperature    treatment efforts tested to date.
      increases in both CAT and luciferase activity. as compared with           Non-ionizing EMF induction of hsp70 is safe, efficient and
      identical transfectants that were sham exposed (Lin et aI.,               practical. These methods can be administered and
      2001).                                                                    readministered prior to and during coronary interventions.
          Interaction with electrons could account for activation of            Furthermore. hsp70 levels can be increased repeatedly with
      DNA by both low and high frequency EMFs. An EMF sensitive                 EMF versus the limited single use of the thermal method.
      DNA sequence suggests that EMFs may interact both directly
      and indirectly with DNA. The initial interaction could involve
      the displacement of electrons in the H-bonds that hold DNA                Summary              and Conclusion
      together, thereby causing chain separation and initiating
                                                                                In these experiments, we report a novel non-invasive technique
      transcription and translation. Blank and Goodman (2007). using
                                                                                to increase hsp70 levels using exposure to low energy. low
      experimentally observed processes as links in a causal chain.
                                                                                frequency EMF. While stress proteins in cells and tissues have
      have proposed that DNA activation of transcription is based on
                                                                                been previously utilized as diagnostic markers and prognostic
      EMF's displacement of electrons in DNA by the EMF and that
                                                                                indicators, a safe, non-invasive method of augmenting
      this causes transient charging of small groups of base pairs (e.g.,
                                                                                endogenous defense mechanisms as a therapeutic tool, such as
      nCTCT n). At the charged sites disaggregation forces overcome
                                                                                EMF exposure, has significant clinical potential. Our data
      the H-bonds, and this disaggregation of the two chains at those
                                                                                indicate that pre-exposure with EMF prior to ischemia and
      sites permits transcription.
                                                                                reperfusion. in a mammalian model, induces upregulation of the
                                                                                HSP70 gene. subsequently increased levels of hsp70 protein,
      Clinical use of EMF technology                                            and, most importantly, improved ventricular function after
                                                                                ischemia-reperfusion.
      An abundance of hsp70 is clearly important to limit myocardial
      injury, following coronary occlusion by reducing infarct size and
      by increasing contractile function. Modulation of hsp70 levels in         Acknowledgments
      the heart, using heat stimulation, is currently problematic from
      a temporal standpoint. It is known that a twofold induction of            This work was partially supported by National Institute of
      hsp70 improves heart muscle cell resistance to oxidation,                 Health Grant T32-HL07854 (I.G.) and the Robert I. Goodman
      ischemia and hypoxia (Heads et al.. 1995; Mestril et aI., 1996;           Fund (R.G.). We would also like to acknowledge Eve Vagg for
      Chong et aI., 1998). Endogenous hsp70 doubles after only I h              her invaluable assistance with the figures.
      following coronary artery occlusion (Loncar et aI., 1998). It may
      take up to 24 h for hsp70 levels to reach a four- to fivefold level.
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