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Commentary

Noninvasive Measurement of EKG Properties of 3D Artificial Heart Muscle

  • Developing and testing a custom fabricated 16-electrode noninvasive direct contact system was necessary to assess the electrical properties of bioengineered heart muscle and to further evaluate the efficacy of cardiac constructs. By culturing neonatal rat primary cardiac cells on a fibrin gel, we constructed 3D artificial heart muscle (3D-AHM), as described in previous studies, which were used in validating this novel system. Electrical and mechanical functional assessment of the tissues was performed, which yielded contractile forces of the tissues, electrical field potential characteristics, and tissue conduction velocities (CV) (20–170 cm/s). Immunohistological evaluation revealed the formation of cardiac tissue structures and cardiomyocyte proliferation. EKG data analysis also yielded time delays between signals in the range of 0–38 ms with electrical maps showing some evidence of synchronous contraction within the fabricated tissues. This study demonstrates the effectiveness and practicality of our novel EKG measuring system to acquire distinct electrical metrics of 3D-AHM, which will aid in increasing the viability and applicability of cardiac tissue constructs.

    Citation: Betsy H. Salazar, Kristopher A. Hoffman, Anilkumar K. Reddy, Sridhar Madala, Ravi K. Birla. Noninvasive Measurement of EKG Properties of 3D Artificial Heart Muscle[J]. AIMS Cell and Tissue Engineering, 2017, 1(1): 12-30. doi: 10.3934/celltissue.2017.1.12

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  • Developing and testing a custom fabricated 16-electrode noninvasive direct contact system was necessary to assess the electrical properties of bioengineered heart muscle and to further evaluate the efficacy of cardiac constructs. By culturing neonatal rat primary cardiac cells on a fibrin gel, we constructed 3D artificial heart muscle (3D-AHM), as described in previous studies, which were used in validating this novel system. Electrical and mechanical functional assessment of the tissues was performed, which yielded contractile forces of the tissues, electrical field potential characteristics, and tissue conduction velocities (CV) (20–170 cm/s). Immunohistological evaluation revealed the formation of cardiac tissue structures and cardiomyocyte proliferation. EKG data analysis also yielded time delays between signals in the range of 0–38 ms with electrical maps showing some evidence of synchronous contraction within the fabricated tissues. This study demonstrates the effectiveness and practicality of our novel EKG measuring system to acquire distinct electrical metrics of 3D-AHM, which will aid in increasing the viability and applicability of cardiac tissue constructs.


    Our goal of this paper is to consider the existence of nodal solution and ground state solution for the following fractional Kirchhoff equation:

    {(a+bu2K)LKu+V(x)u=|u|2α2u+kf(x,u),xΩ,u=0,xR3Ω, (1)

    where a,b,k are positive real numbers. Let α(34,1) such that 2α=632α(4,6) and V(x)C(R3,R+), ΩR3 is a bounded domain with a smooth boundary Ω. u2K:=R3R3|u(x)u(y)|2K(xy)dxdy. The non-local integrodifferential operator LK is defined as follows:

    LKu(x)=12R3(u(x+y)+u(xy)2u(x))K(y)dy,xR3,

    the kernel K:R3(0,) is a function with the properties that

    (ⅰ) mKL1(R3), where m(x)=min{|x|2,1};

    (ⅱ) there exists λ>0 such that K(x)λ|x|(3+2α) for any xR3{0};

    (ⅲ) K(x)=K(x) for any xR3{0}.

    We note that when K(x)=1|x|3+2α, the integrodifferential operator LK is the fractional Laplacian operator (Δ)α:

    (Δ)αu(x)=C(α)2R3(u(x+y)+u(xy)2u(x))|y|3+2αdy

    and in this case

    R3R3|u(x)u(y)|2K(xy)dxdy=2C(α)R3|(Δ)α/2u(x)|2dx,

    where C(α)=(R31cosξ1|ξ|3+2αdξ)1 is the normalized constant.

    When a=1, b=0 and K(x)=1|x|3+2α, the fractional Kirchhoff equation is reduced to the undermentioned fractional nonlocal problem

    {(Δ)αu+V(x)u=|u|2α2u+kf(x,u),xΩ,u=0,xR3Ω. (2)

    Equation (2) is derived from the fractional Schrödinger equation and the nonlinearity f(x,u) represents the particles interacting with each other. On the other hand, recently a great attention has been given to the so called fractional Kirchhoff equation (see [3,10] etc., ):

    (a+bR3R3|u(x)u(y)|2|xy|3+2αdxdy)(Δ)αu=f(x,u), (3)

    where ΩRN is a bounded domain or Ω=RN, a>0,b>0 and u satisfies some boundary conditions. Problem (3) is called to the stationary state of the fractional Kirchhoff equation

    utt+(a+bR3R3|u(x)u(y)|2|xy|3+2αdxdy)(Δ)αu=f(x,u). (4)

    As a special significant case, the nonlocal aspect of the tension arises from nonlocal measurements of the fractional length of the string. For more mathematical and physical background on Schrödinger-Kirchhoff type problems, we refer the readers to [6] and the references therein.

    In the remarkable work of Caffarelli and Silvestre [2], the authors express this nonlocal operator (Δ)α as a Dirichlet-Neumann map for a certain elliptic boundary value problem with local differential operators defined on the upper half space. This technique is a valid tool to deal with the equations involving fractional operator in the respects of regularity and variational methods. However, we do not know whether the Caffarelli-Silvestre extension method can be applied to the general integro-differential operator LK. So the nonlocal feature of the integro-differential operator brings some difficulties to applications of variational method to problem (1). Some of these additional difficulties were overcome in [12,13]. More studies on this integro-differential operator equation, for examples, positive, negative, sign-changing or ground state solutions, we refer the papers [4,5,9,16] and their references. In [1], the authors considered the obstacle problems for LK about the higher regularity of free boundaries. The obstacle problems for nonlinear integro-differential operators and applications are also available in papers [7,8]. The appearance of nonlocal term not only makes it playing an important role in many physical applications, but also brings some difficulties and challenges in mathematical analysis. This fact makes the study of fractional Kirchhoff equation or similar problems particularly interesting. A lot of interesting results on the existence of nonlocal problems were obtained recently, specially on the existence of positive solutions, multiple solutions, ground states and semiclassical states, for examples, we refer [3,15,19] and the cited references.

    In past few years, some researchers began to search for nodal solutions of Schrödinger type equation with critical growth nonlinearity and have got some interesting results. For example, Zhang [20] considered the following Schrödinger-Poisson system:

    {Δu+u+k(x)ϕu=a(x)|u|p2u+u5,xR3,Δϕ=k(x)u2,xR3, (5)

    where p(4,6), k(x) and a(x) are nonnegative functions. By using variational method, a ground state solution and a nodal solution for problem (5) were obtained.

    Wang [17] studies the following Kirchhoff-type equation:

    {(a+bΩ|u|2dx)Δu=|u|4u+λf(x,u), xΩ,u=0, xΩ, (6)

    where ΩR3 is a bounded domain, λ,a,b>0 are fixed parameters, and f(x,) is continuously differentiable for a.e. xΩ. By combining constrained variational method with the degree theory, the existence of ground state and nodal solutions for above problem were obtained.

    However, as for fractional Kirchhoff types equation, to the best of our knowledge, few results involved the existence and asymptotic behavior of ground state and nodal solutions in case of critical growth. If k(x)1, the method used in [20] seems not valid for problem (1), because their result depends on the case kLp(R3)L(R3) for some p(1,2α). We employ minimization arguments on suitable nodal Nehari manifold Mk to construct a nodal solution by using various constrained method and qualitative deformation lemma given in [14].

    It's worth noting that, the Brouwer degree method used in [18] strictly depends on the nonlinearity fC1(R3×R,R), so we have to find new tricks to solve our modeling where we only allow f:R3×RR is a Carathéodory function. On the other hand, in our modeling, both of the nonlocal fractional operator LK and nonlocal terms R3R3|u(x)u(y)|2K(xy)dxdy appear, we need to overcome the difficulties caused by the nonlocal terms under a suitable variational framework. In brief, our discussion is based on the standard nodal Nehari manifold method used in [14].

    Throughout this paper, we let

    E={uX:R3V(x)u2dx<,u=0a.e.inR3Ω},

    where the space X introduced by Servadei and Valdinoci ([12,13]) denotes the linear space of Lebesgue measurable functions u:R3R such that its restriction to Ω u|ΩL2(Ω) and

    ((x,y)(u(x)u(y))K(xy)L2((R3×R3)(Ωc×Ωc),dxdy)

    with the following norm

    ||u||2X=||u||2L2+Q|u(x)u(y)|2K(xy)dxdy,

    where Q=(R3×R3)(Ωc×Ωc). Then, E is a Hilbert space with inner product

    u,v=a2R3R3(u(x)u(y))(v(x)v(y))K(xy)dxdy+ΩV(x)uvdx,u,vE

    and the norm defined by

    u2=a2R3R3|u(x)u(y)|2K(xy)dxdy+ΩV(x)u2dx.

    X0={uX:u=0a.e.inR3Ω} is a Hilbert space with inner product (u,v)K=12R3R3(u(x)u(y))(v(x)v(y))K(xy)dxdy and the Gagliardo norm u2K:=(u,u)K=12R3R3|u(x)u(y)|2K(xy)dxdy. In X0 the norms K and X are equivalent. Thus due to V(x)0 in Ω, the inner product , and the norm defined above still make sense for the Hilbert space E. Moreover, u2Kau2. For more details, we refer to [12].

    The following result for the space X0 will be used repeatedly.

    Lemma 1.1. ([12]) Let s(0,1), N>2s, Ω be an open bounded set of RN, X0↪↪Lp(Ω) for 2<p<2α, and X0L2α(Ω) is continuous. Then, EX0 has the same embedding properties.

    A weak solution uE of (1) is defined to satisfy the following equation

    12(a+bu2K)R3R3(u(x)u(y))(v(x)v(y))K(xy)dxdy+ΩV(x)u(x)v(x)dxΩ|u(x)|2α2u(x)v(x)dxkΩf(x,u(x))v(x)dx=0,

    for any vE, i.e.

    u,v+bu2K(u,v)KkΩf(x,u)vdxΩ|u|2α2uvdx=0,vE.

    As for the function f, we assume f:Ω×RR is a Carathéodory function and satisfing the following hypotheses:

    (f1): f(x,t)t>0 for t0,xΩ and limt0f(x,t)t=0forxΩuniformly;

    (f2): There exists q(4,2α) such that limtf(x,t)tq1=0 for all tR{0} and for xΩ uniformly;

    (f3): f(x,t)|t|3 is an nondecreasing function with respect to t in (,0) and (0,+) for a.e. xΩ.

    Remark 1. We note that under the conditions (f1)-(f3), it is easy to see the function f(x,t)=t3 is an example satisfying all conditions (f1)-(f3).

    The main results can be stated as follows.

    Theorem 1.2. Suppose that (f1)-(f3) are satisfied. Then, there exists k>0 such that for all kk, the problem (1) has a ground state nodal solution uk.

    Remark 2. The ground state nodal solution uk with u±k0 is a solution of (1) satisfying

    Jk(uk)=ck:=infuMkJk(u),

    where Mk is defined by (7) in next section. For uE, u± is defined by

    u+=max{u(x),0},u=min{u(x),0}.

    We recall that the nodal set of a continuous function u:R3R is the set u1(0). Every connected component of R3u1(0) is called a nodal domain. For this equation talking about the nodal domain of uk in somewhat is difficult for us, we leave it as future topics.

    Theorem 1.3. Suppose that (f1)-(f3) are satisfied. Then, there exists k>0 such that for all kk, the c=infuNkJk(u)>0 is achieved by vk, which is a ground state solution of (1) and

    Jk(uk)>2c,

    where Nk={uE {0}|Jk(u),u=0}, uk is the ground state nodal solution obtained in Theorem 1.1.

    Comparing with the literature, the above two results can be regarded as a supplementary of those in [3,4,14,17].

    The remainder of this paper is organized as follows. In Section 2, we give some useful preliminaries. In Section 3, we study the existence of ground state and nodal solutions of (1) and we prove Theorems 1.1-1.2.

    We define the energy functional associated with equation (1) as follows:

    Jk(u)=12u2+b4u4KkΩF(x,u)dx12αΩ|u|2αdx,uE.

    According to our assumptions on f(x,t), Jk(u) belongs to C1(E,R) (see [10,12]), then by direct computations, we have

    Jk(u),v=u,v+bu2K(u,v)KkΩf(x,u)vdxΩ|u|2α2uvdx,u,vE.

    Note that, since (1) involves pure critical nonlinearity |u|2α2u, it will prevent us using the standard arguments as in [14]. Hence, we need some tricks to overcome the lack of compactness of EL2α(R3).

    For fixed uE with u±0, the function φu:[0,)×[0,)R is well defined by φu(s,t)=Jk(su++tu). We set

    H(s,t)=Jk(su++tu),su+,G(s,t)=Jk(su++tu),tu.

    The argument generally used is to modify the method developed in [11]. The nodal Nehari manifold is defined by

    Mk={uE,u±0andJk(u),u+=Jk(u),u=0}, (7)

    which is a subset of the Nehari manifold Nk and contains all nodal solutions of (1). Then, one needs to show the minimizer uMk is a critical point of Jk. Because the problem (1) contains a nonlocal term u2K, the corresponding functional Jk does not have the decomposition

    Jk(u)=Jk(u+)+Jk(u),

    it brings difficulties to construct a nodal solution.

    The following result describes the shape of the nodal Nehari manifold Mk.

    Lemma 2.1. Assume that (f1)-(f3) are satisfied, if uE with u±0, then there is a unique pair (su,tu)(0,)×(0,) such that suu++tuuMk, which is also the unique maximum point of φu on [0,)×[0,). Furthermore, if Jk(u),u±0, then 0<su,tu1.

    Proof. From (f1) and (f2), for any ε>0, there is Cε>0 satisfying

    |f(x,t)|ε|t|+Cε|t|q1,tR. (8)

    By above equality and Sobolev's embedding theorems, we have

    H(s,t):=s2u+2+bsu++tu2K(su++tu,su+)KΩ|su+|2αdxkΩf(x,su+)su+dxast2R3R3[u+(x)u(y)+u(x)u+(y)]K(xy)dxdys2u+2C1s2αu+2αkεC2s2u+2kCεC3squ+q. (9)

    By choosing ε>0 small enough, we can deduce H(s,t)>0 for 0<s1 and all t0. Similarly,

    G(s,t):=t2u2+bsu++tu2K(su++tu,tu)KΩ|tu|2αdxkΩf(x,tu)tudxast2R3R3[u+(x)u(y)+u(x)u+(y)]K(xy)dxdy>0, (10)

    for 0<t1 and all s0. We denote D(u)=u+,u=a(u+,u)K=a2R3R3[u+(x)u(y)+u(x)u+(y)]K(xy)dxdy0. Hence, by choosing δ1>0 small, we have

    H(δ1,t)>0,G(s,δ1)>0. (11)

    For any δ2>δ1, by condition (f1), it is easy to see

    H(δ2,t)δ22u+2+bδ2u++tu2K(δ2u++tu,δ2u+)Kδ22αR3|u+|2αdx+δ2tD(u).

    Similarly, we have

    G(s,δ2)δ22u2+bsu++δ2u2K(su++δ2u,δ2u)Kδ22αR3|u|2αdx+sδ2D(u).

    By choosing δ21, we deduce

    H(δ2,t)<0,G(s,δ2)<0 (12)

    for all s,t[δ1,δ2].

    Following (11) and (12), we can use Miranda's Theorem (see Lemma 2.4 in [17]) to get a positive pair (su,tu)(0,)×(0,) such that suu++tuuMk. Similar to the standard argument in [17], we can prove the pair (su,tu) is unique maximum point of φu on [0,+)×[0,+).

    Lastly, we will prove that 0<su,tu1 when Jk(u),u±0. Following from sutu>0, by a direct computation, we have

    s2uu+2+s2uD(u)+s4ub(u+2K+u2K+2D(u))(u+2K+D(u))s2uu+2+sutuD(u)+b(s2uu+2K+t2uu2K+2sutuD(u))(s2uu+2K+sutuD(u))=s2αuR3|u+|2αdx+kR3f(x,suu+)suu+dx. (13)

    On the other hand, Jk(u),u+0 implies that

    u+2+D(u)+b(u+2K+u2K+2D(u))(u+2K+D(u))R3|u+|2αdx+kR3f(x,u+)u+dx. (14)

    From (13) - (14), we can see that

    (1s2u1)(u+2+D(u))(s2α4u1)R3|u+|2αdx+kR3[f(x,suu+)(suu+)3f(x,u+)(u+)3](u+)4dx.

    So we have su1, which implies that 0<su,tu1.

    Lemma 2.2. There exists k>0 such that for all kk, the infimum ck=infuMkJk(u) is achieved.

    Proof. For any uMk, in view of the definitions of Mk, it follows that

    u±2+bu2K(u,u±)K=kR3f(x,u±)u±dx+R3|u±|2αdx.

    Hence, in view of (8), we have

    u±2kεC1u±2+kC2u±q+C3u±2α.

    By choosing ε>0 small enough, we can deduce

    u±ρ (15)

    for some ρ>0. From assumption (f3), we have

    f(x,t)t4F(x,t)0, (16)

    and f(x,t)t4F(x,t) is nondecreasing in (0,+) and non-increasing in (,0) with respect to the variable t. Hence, combining with Jk(u),u=0, we have

    Jk(u)=14u2+(1412α)R3|u|2αdx+k4R3[f(x,u)u4F(x,u)]dx14u2.

    So ck=infuMkJk(u)0 is well defined.

    Let uE with u±0 be fixed. According to Lemma 2.1, for each k>0, there exists sk,tk>0 such that sku++tkuMk. Hence, by (f1) and the Sobolev's embedding theorem, we have

    s2αkR3|u+|2αdx+t2αkR3|u|2αdxsku++tku2+bsku++tku4K2s2ku+2+2t2ku2+4bs4ku+4K+4bt4ku4K,

    which implies (sk,tk) is bounded and furthermore by taking any sequence (skn,tkn)(s0,t0) as kn, we claim that s0=t0=0. In fact, if s0>0 or t0>0. Thanks to sknu++tknuMkn, we get

    sknu++tknu2+bsknu++tknu4K=R3|sknu++tknu|2αdx+knR3f(sknu++tknu)(sknu++tknu)dx. (17)

    Because kn and {sknu++tknu} is bounded in E, we have a contradiction with the equality (17). On the other hand, we have

    0ckJk(sku++tku)s2ku+2+t2ku2+2bs4ku+4K+2bt4ku4K,

    so limkck=0. By the definition of ck, we can find a sequence {un}Mk satisfing limnJk(un)=ck, which converges to uk=u++uE. By standard arguments, we have

    u±nu±inLp(R3)p(2,2α),u±n(x)u±(x)a.e.xR3.

    Denote β:=s3(S)32s, where

    S:=infuE{0}u2(R3|u|2αdx)22α.

    Sobolev embedding theorem insures that β>0. So that there exists k>0 such that ck<β for all kk. Fix kk, in view of Lemma 2.1, we have Jk(su+n+tun)Jk(un).

    By u±nu±inE, we have

    u±n2u±nu±2=2u±n,u±u±2.

    By taking n in both sides of above equality, there holds

    limnu±n2=limnu±nu±2+u±2.

    On the other hand, by (8) we have

    R3F(x,su±n)dxR3F(x,su±)dx.

    Then,

    lim infnJk(su+n+tun)s22limn(u+nu+2+u+2)+t22limn(unu2+u2)+bs44[limnu+nu+2K+u+2K]2+bt44[limnunu2K+u2K]2+stlim infnD(un)+bs2t24a2lim infnD2(un)+bs2t22lim infn(u+n2Kun2K)+bs3t2alim infn(D(un)u+n2K)+bst32alim infn(D(un)un2K)s2α2αlimn(|u+nu+|2α2α+|u+|2α2α)t2α2αlimn(|unu|2α2α+|u|2α2α)kR3F(x,su+)dxkR3F(x,tu)dx,

    where ||2 and ||2α are the norm in L2(R3) and L2α(R3) repeatedly. So, Fatou's Lemma follows that

    lim infnJk(su+n+tun)Jk(su++tu)+s22limnu+nu+2+t22limnunu2s2α2αlimn|u+nu+|2α2αt2α2αlimn|unu|2α2α+bs42limnu+nu+2Ku+2K+bs44(limnu+nu+2K)2+bt42limnunu2Ku2K+bt44(limnunu2K)2=Jk(su++tu)+s22A1s2α2αB1+t22A2t2α2αB2+bs42A3u+2K+bs44A23+bt42A4u2K+bt44A24,

    where

    A1=limnu+nu+2,A2=limnunu2,B1=limn|u+nu+|2α2α,B2=limn|unu|2α2α,A3=limnu+nu+2K,A4=limnunu2K.

    From the inequality above, we deduce that

    Jk(su++tu)+s22A1s2α2αB1+t22A2t2α2αB2+bs42A3u+2K+bs44A23+bt42A4u2K+bt44A24ck. (18)

    We next prove u±0. Because the claim u0 is similar, so we only prove u+0. Indirectly, we suppose that u+=0 and so A1ρ from (15). By letting t=0 in (18), the case B1=0 is done. So we only study the case B1>0 at length. In this case, by the definition of S, we deduce

    β=α3S32αα3(A1(B1)22α)32α.

    It happens that,

    α3(A1(B1)22α)32α=maxs0{s22A1s2α2αB1}maxs0{s22A1s2α2αB1+bs42A3u+2K+bs44A23}.

    The inequality (18) and ck<β follows that

    βmaxs0{s22A1s2α2αB1+bs42A3u+2K+bs44A23}ck<β,

    which is a contradiction. Thus u+0 are claimed.

    Then, we consider the key point to the proof of Theorem 1.1, that is B1=B2=0 and then ck is achieved by uk=u++uMk.

    Similarly, we only prove B1=0. Indirectly, we suppose that B1>0. We have two cases.

    Case 1: B2>0. Let ˉs and ˉt be the numbers such that

    ˉs22A1ˉs2α2αB1+bˉs42A3u+2K+bˉs44A23=maxs0{s22A1s2α2αB1+bs42A3u+2K+bs44A23},
    ˉt22A2ˉt2α2αB2+bˉt42A4u2K+bˉt44A24=maxt0{t22A2t2α2αB2+bt42A4u2K+bt44A24}.

    Since φu is continuous, we have (su,tu)[0,ˉs]×[0,ˉt] satisfying

    φu(su,tu)=max(s,t)[0,ˉs]×[0,ˉt]φu(s,t).

    If t>0 small enough, then, φu(s,0)<Jk(su+)+Jk(tu)φu(s,t) for all s[0,ˉs]. Thus there is t0[0,ˉt] such that φu(s,0)φu(s,t0) for all s[0,ˉs]. Thus, (su,tu)[0,ˉs]×{0}. Similarly, (su,tu){0}×[0,ˉt].

    By direct computation, we get

    s22A1s2α2αB1+bs42A3u+2K+bs44A23>0, (19)
    t22A2t2α2αB2+bt42A4u2K+bt44A24>0, (20)

    for all (s,t)(0,ˉs]×(0,ˉt]. Hence, there holds

    βˉs22A1ˉs2α2αB1+bˉs42A3u+2K+bˉs44A23+t22A2t2α2αB2+bt42A4u2K+bt44A24,
    βˉt22A2ˉt2α2αB2+bs42A3u+2K+bs44A23+s22A1s2α2αB1+bˉt42A4u2K+bˉt44A24.

    In view of (18), it follows that φu(s,ˉt)0s[0,ˉs] and φu(ˉs,t)0t[0,ˉt]. That is, (su,tu){ˉs}×[0,ˉt] and (su,tu)[0,ˉs]×{ˉt}. Hence, we can deduce that (su,tu)(0,ˉs)×(0,ˉt). By (18), (19) and (20), we deduce

    ckJk(suu++tuu)+s2u2A1s2αu2αB1+t2u2A2t2αu2αB2+bs4u2A3u+2K+bs4u4A23+bt4u2A4u2K+bt4u4A24>Jk(suu++tuu)ck.

    It is impossible. The proof of Case 1 is completed.

    Case 2: B2=0. From the definition of Jk, it is easy to show that there exists t0[0,) such that φu(s,t)0, for all (s,t)[0,ˉs]×[t0,). Thus, there is (su,tu)[0,ˉs]×[0,) satisfying

    φu(su,tu)=max(s,t)[0,ˉs]×[0,)φu(s,t).

    We need to prove that (su,tu)(0,ˉs)×(0,). Similarly, it is noticed that φu(s,0)<φu(s,t) for s[0,ˉs] and 0<t1, that is (su,tu)[0,ˉs]×{0}. Also, for s small enough, we get φu(0,t)<φu(s,t) for t[0,), that is (su,tu){0}×[0,). We note that

    βˉs22A1ˉs2α2αB1+bˉs42A3u+2K+bˉs44A23+t22A2t2α2αB2+bt42A4u2K+bt44A24.

    Thus also from (20) and B2=0, we have φu(ˉs,t)0 for all t[0,). Hence, (su,tu){ˉs}×[0,). That is, (su,tu) is an inner maximizer of φu in [0,ˉs)×[0,). Hence by using (19), we obtain

    ckJk(suu++tuu)+s2u2A1s2αu2αB1+t2u2A2t2αu2αB2+bs4u2A3u+2K+bs4u4A23+bt4u2A4u2K+bt4u4A24>Jk(suu++tuu)ck,

    which is a contradiction.

    Since u±0, by Lemma 2.1, there are su,tu>0 such that ˜u:=suu++tuuMk. On the other hand, Fatou's Lemma follows that

    Jk(u),u±lim infnu±n2+blim infnun2K(un,u±n)KlimnR3f(x,u±n)u±ndxlimnR3|u±n|2α+lim infnL(un)limnJk(un),u±n=0.

    By Lemma 2.1, we know 0<su,tu1. Since unMk and B1=B2=0, we have

    ckJk(˜u)14Jk(˜u),˜u=14(suu+2+tuu2)+(1412α)(|suu+|2α2α+|tuu|2α2α)+k4R3[f(x,suu+)(suu+)4F(x,suu+)]dx+k4R3[f(x,tuu)(tuu)4F(x,tuu)]dxlim infn[Jk(un)14Jk(un),un]=ck.

    So, we have completed proof of Lemma 2.2.

    In this section, we will prove main results.

    Proof. Since ukMk and Jk(u+k+uk)=ck, by Lemma 2.1, for (s,t)(R+×R+)(1,1), we have

    Jk(su+k+tuk)<ck. (21)

    If Jk(uk)0, then there exist δ>0 and θ>0 such that

    Jk(v)θ,forallvuk3δ.

    We know by the result (15), if uMk, there exists L>0 such that u±>L and we can assume 6δ<L. Let Q:=(12,32)×(12,32), and g(s,t)=su+k+tuk, (s,t)Q. In view of (21), it is easy to see that

    ¯ck:=maxQIg<ck. (22)

    Let ε:=min{(ck¯ck)/4,θδ/8} and Sδ:=B(uk,δ), there exists a deformation ηC([0,1]×E,E) satisfying

    (a)] η(t,v)=v if t=0, or v(Jk)1([ck2ε,ck+2ε])S2δ;

    (b)] η(1,(Jk)ck+εSδ)(Jk)ckε;

    (c) ] J _{ k}(\eta(1,v))\leq J _{ k}(v) for all v\in E ;

    (d) ] J _{ k}(\eta(\cdot,v)) is non increasing for every v\in E .

    To finish the proof of Theorem 1.1, one of the key points is to prove that

    \begin{align} \max\limits_{(s,t)\in\bar{Q}}J _{ k}(\eta(1,g(s,t))) < c_k. \end{align} (23)

    The other is to prove that \eta(1,g(Q))\cap\mathcal{M}_k\neq\emptyset . Let us begin this work. In fact, it follows from Lemma 2.1 that g(s,t)\in (J _{ k})^{c_k+\varepsilon}. On the other hand, from (a) and (d), we get

    \begin{align} J _{ k}(\eta(1,v))\leq J _{ k}(\eta(0,v)) = J _{ k}(v),\;\;\forall v\in E. \end{align} (24)

    For (s,t)\in Q , when s\neq 1 or t\neq 1 , according to (21) and (24),

    \begin{align*} J _{ k}(\eta(1,g(s,t)))\leq J _{ k}(g(s,t)) < c_k. \end{align*}

    If s = 1 and t = 1 , that is, g(1,1) = u_k , so that it holds g(1,1)\in J _{ k}^{c_k+\varepsilon}\cap S_{\delta} , then by (b)

    \begin{align*} J _{ k}(\eta(1,g(1,1)))\leq c_k-\varepsilon < c_k. \end{align*}

    Thus (23) holds. Then, let \varphi(s,t): = \eta(1,g(s,t)) and

    \begin{align*} \Upsilon(s,t): = (\frac{1}{s}\langle J'_k(\varphi(s,t)),(\varphi(s,t))^{^{+}}\rangle, \frac{1}{t}\langle J'_k(\varphi(s,t)),(\varphi(s,t))^{^{-}}\rangle). \end{align*}

    The claim holds if there exists (s_{0},t_{0})\in Q such that \Upsilon(s_{0},t_{0}) = (0,0) . Since

    \begin{align*} \|g(s,t)-u_k\|^{2} = &\|(s-1)u_k^{+}+(t-1)u_k^{-}\|^{2}\\ \geq&|s-1|^2\|u_k^{+}\|^{2} > |s-1|^2(6\delta)^{2}, \end{align*}

    and |s-1|^2(6\delta)^{2}>4\delta^{2}\Leftrightarrow s<2/3\;\;\rm{or}\;\;s>4/3 , using (ⅰ) and the range of s , for s = \frac{1}{2} and for every t\in[\frac{1}{2},\frac{3}{2}] we have g(\frac{1}{2},t)\notin S_{2\delta} , so from (a), we have \varphi(\frac{1}{2},t) = g(\frac{1}{2},t) . Thus

    \begin{align*} \Upsilon(\frac{1}{2},t) = \large(2\langle J'_k(\frac{1}{2}u_k^{+}+t u_k^{-}),\frac{1}{2}u_k^{+}\rangle,\frac 1t\langle J _{ k}'(\frac{1}{2}u_k^{+}+t u_k^{-}),tu^{-}\rangle\large). \end{align*}

    On the other hand, from (9) and u\in \mathcal{M} _{k} , we have that

    \begin{align*} H(t,t) = &(t^{2}-t^4)(\|u^{+}\|^{2}+D(u))+(t^{4}-t^{2_\alpha^*}) \int_{\mathbb{R}^{3}}|u^{+}|^{2^{\ast}_{\alpha}}dx\\ &+kt^4\int_{\mathbb{R}^{3}}\left(f(x,u^+)- \frac{f(x,tu^{+})}{t^3}\right) u^{+}dx. \end{align*}

    According to (f_{3}) , when 0<t<1 , H(t,t)>0, while in the case t>1 , H(t,t)<0. Similarly, it is easy to get G(t,t)>0\;\rm{for}\;t\in(0,1),\;\; G(t,t)<0\;\rm{for}\;t>1. By above discussions, due to (f_{3}) and 2_\alpha^*>4 , we have

    \begin{align*} H(\frac{1}{2},t) = &\|\frac{1}{2}u_k^{+}\|^2+\frac{t}{2}D(u_k)\\ &+b\big(\frac{1}{4}\|u_k^{+}\|_{K}^2+t^2\|u_k^{-}\|_{K}^2+ \frac taD(u_k)\big)\big(\frac{1}{4}\|u_k^{+}\|_{K}^2+\frac{t}{2a}D(u_k)\big)\\ &-(\frac{1}{2})^{2^{\ast}_{\alpha}}\int_{\mathbb{R}^{3}}|u_k^{+}|^{2^{\ast}_{\alpha}}dx - k\int_{\mathbb{R}^{3}}f(x,\frac{1}{2}u_k^{+})\frac{1}{2}u_k^{+}dx\geq H(\frac 12, \frac 12) > 0, \end{align*}

    which implies that

    \begin{align} H(\frac{1}{2},t) > 0,\;\;\forall t\in[\frac{1}{2},\frac{3}{2}]. \end{align} (25)

    Analogously, \varphi(\frac{3}{2},t) = g(\frac{3}{2},t) implies that

    \begin{align*} H(\frac{3}{2},t) = &\|\frac{3}{2}u_k^{+}\|^2+\frac{3t}{2}D(u_k) +b\big(\frac{9}{4}\|u_k^{+}\|_{K}^2+t^2\|u_k^{-}\|_{K}^2\\ &+\frac{3t}aD(u_k)\big)\big(\frac{9}{4}\|u_k^{+}\|_{K}^2+\frac{3t}{2a}D(u_k)\big)\\ &-(\frac{3}{2})^{2^{\ast}_{\alpha}}\int_{\mathbb{R}^{3}}|u_k^{+}|^{2^{\ast}_{\alpha}}dx - k\int_{\mathbb{R}^{3}}f(x,\frac{3}{2}u_k^{+})\frac{3}{2}u_k^{+}dx\leq H(\frac{3}{2},\frac{3}{2}) < 0, \end{align*}

    that is,

    \begin{align} H(\frac{3}{2},t) < 0,\;\;\forall t\in[\frac{1}{2},\frac{3}{2}]. \end{align} (26)

    By the same way,

    \begin{align} G(s,\frac{1}{2}) > 0,\;\;\forall s\in[\frac{1}{2},\frac{3}{2}],\;\;\rm{and}\;\; G(s,\frac{3}{2}) > 0,\;\;\forall s\in[\frac{1}{2},\frac{3}{2}]. \end{align} (27)

    From (25)-(27), the assumptions of Miranda's Theorem (see Lemma 2.4 in [17]) are satisfied. Thus, there exists (s_{0},t_{0})\in Q such taht \Upsilon(s_{0},t_{0}) = 0 , i.e. \eta(1,g(s_{0},t_{0}))\in \mathcal{M}_{ k} . Comparing to (23), u_k is a ground state nodal solution of problem (1).

    Proof. Recall that \beta = \frac{\alpha}{3}S^{\frac{3}{2\alpha}} , where S: = \inf_{u\in E\backslash\{0\}}\frac{\|u\|^{2}}{(\int_{\mathbb{R}^{3}}|u|^{2^{\ast}_{\alpha}}dx)^{\frac{2}{2^{\ast}_{\alpha}}}}. Similar to the proof of Lemma 2.2, we claim that there exists k_{1}^{\star}>0 such that for all k\geq k_{1}^{\star} , there exists v_k\in \mathcal{N}_k such that J _{ k}(v_k) = c^{\ast}>0 and there is k^{\star}>0 such that c^{\ast}<\beta for all k\geq k^{\star} . By standard processes, we can assume v_{n}\rightharpoonup v_k\in E. Therefore, \liminf_{n\rightarrow \infty}J _{ k}(tv_{n})\geq c^*\geq J _{ k}(tv_k)+\frac{t^{2}}{2}A-\frac{t^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B +\frac{bt^{4}}{4}(C^2+2C\|v_k\|_{K}^2) , where A = \lim_{n\rightarrow \infty}\|v_{n}-v_k\|^{2}, B = \lim_{n\rightarrow \infty}|v_{n}-v_k|_{2^{\ast}_{\alpha}}^{2^{\ast}_{\alpha}} and C = \|v_{n}-v_k\|_{K}^2 .

    Firstly, we prove that v_k\neq0 . By contradiction, we suppose v_k = 0 . The Case B = 0 is contained in above equality. So we only consider the case B>0 . The fact \beta\leq\frac{\alpha}{3}\left(\frac{A_{1}}{(B_{1})^{\frac{2}{2^{\ast}_{\alpha}}}}\right)^{\frac{3}{2\alpha}} follows that

    \begin{align*} \beta\leq&\frac{\tilde{t}^{2}}{2}A-\frac{\tilde{t}^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B : = \max\limits_{t\geq0}\{\frac{t^{2}}{2}A-\frac{t^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B\}\\ \leq&\max\limits_{t\geq0}\{\frac{t^{2}}{2}A-\frac{t^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B +\frac{bt^{4}}{4}(C^2+2C\|v_k\|_{K}^2)\}\le c^* < \beta. \end{align*}

    Which is a contradiction.

    Then, we prove that B = 0 and c^{\ast}\; is\; achieved \; by \; v_k . Indirectly, we suppose that B>0 . Firstly, we can maximize \varphi_{v_k}(t) = J _{ k}(tv_k) in [0,\infty) at t_{v} , which be an inner maximizer. So we get \frac{t_v^{2}}{2}A-\frac{t_v^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B +\frac{bt_v^{4}}{4}(C^2+2C\|v_k\|_{K}^2)>0 . Thus from t_{v}v_k\in \mathcal{N}_{b}^{ k} we get a contradiction by

    c^*\le J _{ k}(t_vv_k) < J _{ k}(t_vv_k)+\frac{t_v^{2}}{2}A-\frac{t_v^{2^{\ast}_{\alpha}}}{2^{\ast}_{\alpha}}B +\frac{bt_v^{4}}{4}(C^2+2C\|v_k\|_{K}^2)\le c^*.

    From the above arguments we know \widetilde{v}: = t_{v}v_k\in \mathcal{N}_k . Furthermore, we have

    \begin{align*} c^*\le& J _{ k}(\tilde v)-\frac 14 \langle J'_k(\tilde{v}),\tilde{v}\rangle \le\frac 14\|v_k\|^2\\ &+(\frac 1{4}-\frac 1{2^{\ast}_{\alpha}})|v_k|_{2^{\ast}_{\alpha}}^{2^{\ast}_{\alpha}}+\frac k4\int_{\mathbb{R}^3}[f(x,v_k)v_k-4F(x,v_k)]dx\\ = &\liminf\limits_{n\to \infty}[J _{ k}(v_n)-\frac 14 \langle J'_k(v_n),v_n\rangle] = c^*. \end{align*}

    Therefore, t_{v} = 1 , and c^{\ast} is achieved by v_k\in \mathcal{N}_k .

    By standard arguments, we can find v_k\in E , a ground state solution of problem (1). For all k\geq k^{\star} , the problem (1) also has a ground state nodal solution u_k . Let k^{\star\star} = \max\{ k^{\star}, k^{\star}_{1}\}. Suppose that u_k = u^{+}+u^{-} , we let s_{u^{+}},t_{u^{-}}\in(0,1) such that

    s_{u^{+}}u^{+}\in \mathcal{N}_k,\;\;\; t_{u^{-}}u^{-}\in \mathcal{N}_k.

    Thus, the above fact follows that

    \begin{align*} 2c^{\ast}\leq J _{ k}(s_{u^{+}}u^{+})+J _{ k}(t_{u^{-}}u^{-})\leq J _{ k}(s_{u^{+}}u^{+}+t_{u^{-}}u^{-}) < J _{ k}(u^{+}+u^{-}) = c_k. \end{align*}
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