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(Circulation Research. 2005;96:815.)
© 2005 American Heart Association, Inc.

Report

Adenoviral Gene Transfer of Mutant Phospholamban Rescues Contractile Dysfunction in Failing Rabbit Myocytes With Relatively Preserved SERCA Function

Mark T. Ziolo, Jody L. Martin, Julie Bossuyt, Donald M. Bers, Steven M. Pogwizd

From the Department of Physiology (M.T.Z., J.L.M., J.B., D.M.B.), Loyola University Chicago, Maywood, Ill; and the Department of Medicine (S.M.P.), University of Illinois at Chicago, Chicago, Ill.

Correspondence to Mark T. Ziolo, Department of Physiology and Cell Biology, Ohio State University, 304 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210-1218. E-mail ziolo.1{at}osu.edu

	Abstract

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In heart failure (HF) a main factor in reduced contractility is reduced SR Ca²⁺ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca²⁺-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with ß-galactosidase adenovirus as control) could enhance SERCA function and restore Ca²⁺ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca²⁺ transient amplitude (2.0±0.1 versus 1.6±0.05 F/F_o at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adßgal had negative FFR. Ca²⁺ transients declined faster in AdPLB-dn versus Adßgal myocytes (RT_50%: 317±29 versus 551±90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca²⁺ load (3.5±0.3 versus 2.6±0.2 F/F_o at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca²⁺ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.

Key Words: heart failure • gene transfer • SERCA • phospholamban

	Introduction

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Heart failure (HF) is characterized by a reversed force frequency response (FFR), ie, positive to negative.¹ This has been attributed to alterations in the expression and/or function of the sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA)² and reduced SR Ca²⁺ content.³

Transgenic and adenoviral gene transfer approaches to increase SERCA activity, either by increasing SERCA levels⁴ or decreasing inhibition by phospholamban (PLB; by PLB knockout, PLB antisense, or a pseudophosphorylated PLB mutant)^4–6 can prevent the development of HF^7,8 and increase contractility,^4–6 although mechanistic information was limited.

Not all HF exhibits decreased SERCA expression and/or function, and Hasenfuss et al⁹ showed that nearly half of HF patients exhibit relatively preserved SERCA function but greatly increased Na⁺/Ca²⁺ exchanger (NCX) function. This is analogous to our nonischemic rabbit HF model, where contractile function and SR Ca²⁺ load are reduced mainly because of increased NCX function rather than depressed SERCA function or SR Ca²⁺ leak.^10–12

It is unclear whether enhancement of SERCA function would be beneficial in myocytes during chronic HF and where SERCA is not the main dysfunction. Thus, the purpose of this study was to determine whether (and how) increasing SERCA function, via ectopic expression of a PLB dominant-negative mutant, can reverse contractile dysfunction.

	Materials and Methods

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Rabbit Heart Failure Model and Cardiac Myocyte Isolation
In New Zealand White rabbits, HF was induced by aortic insufficiency and 2 to 4 weeks later by aortic constriction.^10,11 HF progression was assessed by echocardiography and rabbits were studied 7.1±1.9 months after aortic constriction when LV end-systolic dimension exceeded 1.4 cm.¹¹ Left ventricular (LV) myocytes were isolated as previously described.^10,11

Adenoviral Gene Transfer and Cell Culture
HF myocytes were cultured for adenoviral infection in modified M199 medium for 24 to 36 hours after infection. Myocytes were infected with adenovirus (MOI=100 to 200, 1 to 2 hour infection) encoding either ß-galactosidase (Adßgal, served as control) or a dominant-negative PLB mutant [AdPLB-dn, with amino acid substitutions at positions 3 and 14 (K3E/R14E), which inhibits PLB function].¹³

Western Blots
Antibodies for PLB (Badrilla), SERCA (Affinity Bioreagents), and NCX (Affinity Bioregants) were used and data normalized to sarcomeric -actin (Sigma).¹⁰

Measurement of Ca²⁺ Transients
Myocytes were field-stimulated and superfused with (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 5 HEPES (pH=7.4). [Ca²⁺]_i was measured by Fluo-3 epifluorescence with excitation at 480 nm and emission at 530 nm, with recorded fluorescence (F) normalized to baseline fluorescence (F_o).¹⁴

	Results

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HF Rabbits
HF rabbits exhibited significant depression of LV systolic function; with increases in both LV end-diastolic (by 50%) and end-systolic dimension (by 73%; P<0.001) and a 27% decrease in LV fractional shortening (P<0.01 versus baseline).

AdPLB-dn Enhances Ca²⁺ Transients, Force-Frequency Response, and SR Ca²⁺ Load
Cells exposed to AdPLB-dn (versus Adßgal) showed increased PLB expression and the characteristic¹³ decrease in electrophoretic mobility of pentamers in Western blots (Figure 1A). In addition, infection with AdPLB-dn or Adßgal did not alter expression of either NCX or SERCA2a versus acutely isolated HF myocytes (Figure 1B).

View larger version (35K): [in this window] [in a new window]   Figure 1. AdPLB-dn and Ca2+ handling in HF myocytes. A, Western blot of HF rabbit myocytes infected with AdPLB-dn or Adßgal (MOI 100 or 200), showing heteropentamer shift due to mutant PLB, as in ref 13. B, Pooled data of NCX and SERCA expression after infection with AdPLBdn or Adßgal (vs acutely isolated HF myocytes). C, Ca2+ transients in HF myocytes infected with Adßgal or AdPLBdn (0.5 Hz). D, Pooled data for Ca2+ transient amplitude and (E) force-frequency relationship (FFR). Data are mean±SEM. *P<0.05 vs acute and Adßgal (n=6 to 10 cells from 4 HF hearts).

Figure 1C and 1D shows that steady-state Ca²⁺ transient amplitude in HF myocytes was increased by 58% after AdPLB-dn versus Adßgal infection. Notably, Adßgal had similar Ca²⁺ transient amplitude compared with acutely isolated noncultured, noninfected HF myocytes (from the same HF hearts used for adenoviral infection). Figure 1E shows that HF myocytes acutely isolated or infected with Adßgal displayed the negative FFR typical of HF, whereas HF myocytes infected with AdPLB-dn had a positive FFR.

We also assessed whether the enhanced function in AdPLB-dn is due to increased SERCA function and SR Ca²⁺ load (despite unaltered SERCA or NCX expression). The rate constant of [Ca²⁺]_i decline in myocytes infected with AdPLB-dn was 42% faster than that in Adßgal (Figure 2A), consistent with enhanced SERCA function. Likewise, SR Ca²⁺ content was significantly increased in AdPLB-dn versus Adßgal (Figure 2B).

View larger version (17K): [in this window] [in a new window]   Figure 2. AdPLBdn: Ca2+ transient kinetics and SR Ca content in HF myocytes. A, Half-time of [Ca]i decline during twitches and (B) SR Ca2+ load assessed by rapid application of 10 mmol/L caffeine. Pooled data (mean±SEM; *P<0.05 vs Adßgal; n=8 to 10 cells from 4 HF hearts).

	Discussion

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HF myocytes typically demonstrate decreased SR Ca²⁺ load, Ca²⁺ transient amplitude, and a negative FFR.^1,10,12 Although this is frequently attributed to decreased SR Ca²⁺ uptake and SERCA function,^2,3 reduced SR Ca²⁺ load in HF models and human HF can arise from upregulation of the NCX.^9,10 Moreover, adenoviral overexpression of NCX in normal rabbit ventricular myocytes decreased SR Ca²⁺ content and Ca²⁺ transients.¹⁵

In this study, we found that, even after HF development, inhibition of PLB function enhanced Ca²⁺ transient amplitude, rate of [Ca²⁺]_i decline, and SR Ca²⁺ content and restored a positive FFR in LV HF myocytes (without altered NCX or SERCA expression). This HF model exhibits severely depressed LV and myocyte function and reduced SR Ca²⁺ content, 2-fold upregulation of NCX expression and function, enhanced SR Ca²⁺ leak, and unchanged SERCA expression (but detailed quantitative analysis resolved a 24% decrease in SERCA function).^10–12 The main cause of depressed SR Ca²⁺ content was found to be the increased NCX function (not altered SERCA or SR Ca²⁺ leak).¹²

We show, for the first time in HF myocytes, that PLB inhibition increased SR Ca²⁺ load and restored a positive FFR, which can explain most of the beneficial effects observed by us and others.^4–8 This indicates that enhanced SERCA function can overcome the NCX upregulation and functional normalization of Ca²⁺ transients.

As in HF myocytes, nonfailing rabbit ventricular myocytes also showed significantly enhanced Ca²⁺ transient amplitude (by 43%) and rate of [Ca]_i decline (by 20%), as previously reported.¹³ Precisely how this dominant-negative PLB effect is mediated is unclear, but it may either sequester endogenous PLB in pentameric form (Figure 1A) relieving SERCA of inhibition, or displace endogenous PLB from SERCA2 (without inhibiting the pump itself). A quantitative limitation is that we cannot assess the effect of AdPLB-dn on endogenous PLB expression because the antibody used recognizes both endogenous and mutant PLB.

SERCA function in HF can be stimulated by either SERCA overexpression or interfering with the inhibition of SERCA by PLB (PLB knockout or by viral gene transfer of antisense PLB RNA or of a pseudophophorylated form of PLB mutant). These can all improve myocyte and cardiac contractile function and prevent the development or progression of HF, including in some animal models of HF.^4–8 Our data indicates that we are able to restore contractility even long after the onset of HF. In addition, it may be more advantageous to inhibit PLB function versus SERCA overexpression because too much SERCA may increase cytosolic Ca²⁺ buffering and accelerate [Ca]_i decline in a manner that limits myofilament activation.¹⁶ Inhibition of the NCX may also be beneficial,¹⁷ but excessive NCX inhibition may prevent necessary Ca²⁺ removal from the cell. Our results attest to the potential of PLB as a therapeutic target for the treatment of HF even when reduced SERCA activity is not the major abnormality.

In conclusion, enhanced SERCA function via gene transfer of a dominant-negative PLB mutant reversed the contractile dysfunction in HF myocytes by increasing SR Ca²⁺ load and restoring the positive FFR. This may justify future tests of this rescue approach in vivo.

	Acknowledgments

 
This work was supported by NIH grants HL46929 and HL73966 (S.M.P.) and HL64724 and HL30077 (D.M.B.).

	Footnotes

 
Original received October 18, 2004; resubmission received February 25, 2005; revised resubmission received March 15, 2005; accepted March 16, 2005.

	References

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1. Rossman EI, Petre RE, Chaudhary KW, Piacentino V III, Janssen PML, Gaughan JP, Houser SR, Margulies KB. Abnormal frequency-dependent responses represent the pathophysiological signature of contractile failure in human myocardium. J Mol Cell Cardiol. 2004; 36: 33–42.[CrossRef][Medline] [Order article via Infotrieve]

2. Kubo H, Margulies KB, Piacentino V III, Gaughan JP, Houser SR. Patients with end-stage congestive heart failure treated with ß-adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance. Circulation. 2001; 104: 1012–1018.[Abstract/Free Full Text]

3. Piacentino V III, Weber CR, Chen X, Weisser-Thomas J, Margulies KB, Bers DM, Houser SR. Cellular basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651–658.[Abstract/Free Full Text]

4. Del Monte F, Harding SE, Dec GW, Gwathmey JK, Hajjar RJ. Targeting phospholamban by gene transfer. Circulation. 2002; 105: 904–907.[Abstract/Free Full Text]

5. Iwanaga Y, Hoshijima M, Gu Y, Iwatate M, Dieterle T, Ikeda Y, Date MO, Chrast J, Matsuzaki M, Peterson KL, Chien KR, Ross J. Chronic phospholamban inhibition prevents progressive cardiac dysfuncion and pathological remodeling after infarction in rats. J Clin Invest. 2004; 113: 727–736.[CrossRef][Medline] [Order article via Infotrieve]

6. Hoshijima M, Ikeda Y, Iwanaga Y, Minamisawa S, Date M, Gu Y, Iwatate M, Li M, Wang L, Wilson JM, Wang Y, Ross J, Chien KR. Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nature Medicine. 2002; 8: 864–871.[Medline] [Order article via Infotrieve]

7. Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J, Kranias EG, Giles WR, Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell. 1999; 99: 313–322.[CrossRef][Medline] [Order article via Infotrieve]

8. Freeman K, Lerman I, Kranias EG, Bohlmeyer T, Bristow MR, Lefkowitz RJ, Iaccarino G, Koch WJ, Leinwand LA. Alternations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J Clin Invest. 2001; 107: 967–974.[Medline] [Order article via Infotrieve]

9. Hasenfuss G, Schillinger W, Lehnart SE, Preuss M, Pieske B, Maier LS, Prestle J, Minami K, Just H. Relationship between Na⁺-Ca²⁺-exchanger protein levels and diastolic function of failing human myocardium. Circulation. 1999; 99: 641–648.[Abstract/Free Full Text]

10. Pogwizd SM, Qi M, Yuan W, Samarel AM, Bers DM. Upregulation of Na⁺/Ca²⁺ exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res. 1999; 85: 1009–1019.[Abstract/Free Full Text]

11. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current and residual ß-adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.[Abstract/Free Full Text]

12. Shannon TR, Pogwizd SM, Bers DM. Elevated sarcoplasmic reticulum Ca²⁺ leak in intact ventricular myocytes from rabbits in heart failure. Circ Res. 2003; 93: 592–594.[Abstract/Free Full Text]

13. He H, Meyer M, Martin JL, McDonough PM, Ho P, Lou X, Lew WY, Hilal-Dandan R, Dillmann WH. Effects of mutant and antisense RNA of phospholamban on SR Ca²⁺-ATPase activity and cardiac myocyte contractility. Circulation. 1999; 100: 974–980.[Abstract/Free Full Text]

14. Ziolo MT, Maier LS, Piacentino V, III, Bossuyt J, Houser SR, Bers DM. Myocyte NOS2 contributes to blunted ß-adrenergic response in failing human hearts by decreasing Ca²⁺ transients. Circulation. 2004; 109: 1886–1891.[Abstract/Free Full Text]

15. Schillinger W, Janssen PML, Emami S, Henderson SA, Ross RS, Teucher N, Zeitz O, Phillipson KD, Prestle J, Hasenfuss G. Impaired contractile performance of cultured rabbit ventricular myocytes after adenoviral gene transfer of Na⁺-Ca²⁺ exchanger. Circ Res. 2000; 87: 581–587.[Abstract/Free Full Text]

16. Teucher N, Prestle J, Seidler T, Inesi G, Kögler H, Bers DM, Hasenfuss G, Smith GL: Excessive SERCA expression causes increased sarcoplasmic reticulum Ca²⁺-uptake but decreased contractile performance. Circulation. 2004; 110: 3553–3559.[Abstract/Free Full Text]

17. Hobai IA, Maack C, O’Rourke B. Partial inhibition of sodium/calcium exchange restores cellular calcium handling in canine heart failure. Circ Res. 2004; 95: 292–299.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) All Versions of this Article: 96/8/815    most recent 01.RES.0000163981.97262.3bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ziolo, M. T. Articles by Pogwizd, S. M. Search for Related Content PubMed PubMed Citation Articles by Ziolo, M. T. Articles by Pogwizd, S. M. Related Collections Contractile function Congestive Animal models of human disease Calcium cycling/excitation-contraction coupling Heart failure - basic studies Gene therapy