The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase

Abstract

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.

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References

  1. 1.

    Aiello EA, Petroff MG, Mattiazzi AR, Cingolani HE (1998) Evidence for an electrogenic Na+-HCO3 symport in rat cardiac myocytes. J Physiol 512(Pt 1):137–148

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Arabacilar P, Marber M (2015) The case for inhibiting p38 mitogen-activated protein kinase in heart failure. Front Pharmacol 6:102. https://doi.org/10.3389/fphar.2015.00102

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Bassani RA, Bers DM (1994) Na-Ca exchange is required for rest-decay but not for rest-potentiation of twitches in rabbit and rat ventricular myocytes. J Mol Cell Cardiol 26:1335–1347

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313. https://doi.org/10.1152/physrev.00044.2005

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Bers DM (2010) CaMKII inhibition in heart failure makes jump to human. Circ Res 107:1044–1046. https://doi.org/10.1161/CIRCRESAHA.110.231902

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Bers DM, Bassani RA, Bassani JW, Baudet S, Hryshko LV (1993) Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium. J Mol Cell Cardiol 25:1047–1057. https://doi.org/10.1006/jmcc.1993.1117

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Bossuyt J, Helmstadter K, Wu X, Clements-Jewery H, Haworth RS, Avkiran M, Martin JL, Pogwizd SM, Bers DM (2008) Ca2+/calmodulin-dependent protein kinase IIdelta and protein kinase D overexpression reinforce the histone deacetylase 5 redistribution in heart failure. Circ Res 102:695–702. https://doi.org/10.1161/CIRCRESAHA.107.169755

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Caldiz CI, Diaz RG, Nolly MB, Chiappe de Cingolani GE, Ennis IL, Cingolani HE, Perez NG (2011) Mineralocorticoid receptor activation is crucial in the signalling pathway leading to the Anrep effect. J Physiol 589:6051–6061

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Camara AK, Lesnefsky EJ, Stowe DF (2010) Potential therapeutic benefits of strategies directed to mitochondria. Antioxid Redox Signal 13:279–347. https://doi.org/10.1089/ars.2009.2788

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Camilion de Hurtado MC, Alvarez BV, Perez NG, Cingolani HE (1996) Role of an electrogenic Na+-HCO3 cotransport in determining myocardial pHi after an increase in heart rate. Circ Res 79:698–704

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Chen Y, Rajashree R, Liu Q, Hofmann P (2003) Acute p38 MAPK activation decreases force development in ventricular myocytes. Am J Phys Heart Circ Phys 285:H2578–H2586. https://doi.org/10.1152/ajpheart.00365.2003

    CAS  Google Scholar 

  12. 12.

    Cingolani HE, Villa-Abrille MC, Cornelli M, Nolly A, Ennis IL, Garciarena C, Suburo AM, Torbidoni V, Correa MV, Camilionde Hurtado MC, Aiello EA (2006) The positive inotropic effect of angiotensin II: role of endothelin-1 and reactive oxygen species. Hypertension 47:727–734. https://doi.org/10.1161/01.HYP.0000208302.62399.68

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    De Giusti VC, Correa MV, Villa-Abrille MC, Beltrano C, Yeves AM, de Cingolani GE, Cingolani HE, Aiello EA (2008) The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species. Life Sci 83:264–271. https://doi.org/10.1016/j.lfs.2008.06.008

    Article  PubMed  Google Scholar 

  14. 14.

    De Giusti VC, Garciarena CD, Aiello EA (2009) Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3 cotransport. J Mol Cell Cardiol 47:716–722

    Article  PubMed  Google Scholar 

  15. 15.

    De Giusti VC, Nolly MB, Yeves AM, Caldiz CI, Villa-Abrille MC, Chiappe de Cingolani GE, Ennis IL, Cingolani HE, Aiello EA (2011) Aldosterone stimulates the cardiac Na+/H+ exchanger via transactivation of the epidermal growth factor receptor. Hypertension 58:912–919. https://doi.org/10.1161/HYPERTENSIONAHA.111.176024

    Article  PubMed  Google Scholar 

  16. 16.

    De Giusti VC, Orlowski A, Ciancio MC, Espejo MS, Gonano LA, Caldiz CI, Vila Petroff MG, Villa-Abrille MC, Aiello EA (2015) Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30. J Mol Cell Cardiol 89:260–267. https://doi.org/10.1016/j.yjmcc.2015.10.024

    Article  PubMed  Google Scholar 

  17. 17.

    Dedkova EN, Seidlmayer LK, Blatter LA (2013) Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure. J Mol Cell Cardiol 59:41–54. https://doi.org/10.1016/j.yjmcc.2013.01.016

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Dikalov S (2011) Cross talk between mitochondria and NADPH oxidases. Free Radic Biol Med 51:1289–1301. https://doi.org/10.1016/j.freeradbiomed.2011.06.033

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Drose S (2013) Differential effects of complex II on mitochondrial ROS production and their relation to cardioprotective pre- and postconditioning. Biochim Biophys Acta 1827:578–587. https://doi.org/10.1016/j.bbabio.2013.01.004

    Article  PubMed  Google Scholar 

  20. 20.

    Endoh M (2004) Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance. Eur J Pharmacol 500:73–86. https://doi.org/10.1016/j.ejphar.2004.07.013

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Fischer TH, Kleinwachter A, Herting J, Eiringhaus J, Hartmann N, Renner A, Gummert J, Haverich A, Schmitto JD, Sossalla S (2016) Inhibition of CaMKII attenuates progressing disruption of Ca(2+) homeostasis upon left ventricular assist device implantation in human heart failure. Artif Organs 40:719–726. https://doi.org/10.1111/aor.12677

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Greensmith DJ, Eisner DA, Nirmalan M (2010) The effects of hydrogen peroxide on intracellular calcium handling and contractility in the rat ventricular myocyte. Cell Calcium 48:341–351. https://doi.org/10.1016/j.ceca.2010.10.007

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Haworth RS, Dashnyam S, Avkiran M (2006) Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes. Biochem J 399:493–501

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Heinzel FR, Luo Y, Dodoni G, Boengler K, Petrat F, Di Lisa F, de Groot H, Schulz R, Heusch G (2006) Formation of reactive oxygen species at increased contraction frequency in rat cardiomyocytes. Cardiovasc Res 71:374–382. https://doi.org/10.1016/j.cardiores.2006.05.014

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Hoch B, Meyer R, Hetzer R, Krause EG, Karczewski P (1999) Identification and expression of delta-isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human myocardium. Circ Res 84:713–721

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Kimura S, Zhang GX, Nishiyama A, Shokoji T, Yao L, Fan YY, Rahman M, Suzuki T, Maeta H, Abe Y (2005) Role of NADPH oxidase- and mitochondria-derived reactive oxygen species in cardioprotection of ischemic reperfusion injury by angiotensin II. Hypertension 45:860–866. https://doi.org/10.1161/01.HYP.0000163462.98381.7f

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Lakatta EG, DiFrancesco D (2009) What keeps us ticking: a funny current, a calcium clock, or both? J Mol Cell Cardiol 47:157–170. https://doi.org/10.1016/j.yjmcc.2009.03.022

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Liao P, Wang SQ, Wang S, Zheng M, Zheng M, Zhang SJ, Cheng H, Wang Y, Xiao RP (2002) p38 mitogen-activated protein kinase mediates a negative inotropic effect in cardiac myocytes. Circ Res 90:190–196

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Liao X, He J, Ma H, Tao J, Chen W, Leng X, Mai W, Zhen W, Liu J, Wang L (2007) Angiotensin-converting enzyme inhibitor improves force and Ca2+-frequency relationships in myocytes from rats with heart failure. Acta Cardiol 62:157–162. https://doi.org/10.2143/AC.62.2.2020236

    Article  PubMed  Google Scholar 

  30. 30.

    Maier LS, Bers DM (2002) Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond. J Mol Cell Cardiol 34:919–939

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Maier LS, Pieske B, Allen DG (1997) Influence of stimulation frequency on [Na+]i and contractile function in Langendorff-perfused rat heart. Am J Phys 273:H1246–H1254

    CAS  Google Scholar 

  32. 32.

    Maier LS, Brandes R, Pieske B, Bers DM (1998) Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium. Am J Phys 274:H1361–H1370

    CAS  Google Scholar 

  33. 33.

    Maier LS, Bers DM, Pieske B (2000) Differences in Ca2+-handling and sarcoplasmic reticulum Ca2+-content in isolated rat and rabbit myocardium. J Mol Cell Cardiol 32:2249–2258. https://doi.org/10.1006/jmcc.2000.1252

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Mallat Z, Philip I, Lebret M, Chatel D, Maclouf J, Tedgui A (1998) Elevated levels of 8-iso-prostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation 97:1536–1539

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Mills GD, Harris DM, Chen X, Houser SR (2007) Intracellular sodium determines frequency-dependent alterations in contractility in hypertrophied feline ventricular myocytes. Am J Phys Heart Circ Phys 292:H1129–H1138. https://doi.org/10.1152/ajpheart.00375.2006

    CAS  Google Scholar 

  36. 36.

    Morgan PE, Aiello EA, Chiappe de Cingolani GE, Mattiazzi AR, Cingolani HE (1999) Chronic administration of nifedipine induces up-regulation of functional calcium channels in rat myocardium. J Mol Cell Cardiol 31:1873–1883. https://doi.org/10.1006/jmcc.1999.1019

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Morii I, Kihara Y, Konishi T, Inubushi T, Sasayama S (1996) Mechanism of the negative force-frequency relationship in physiologically intact rat ventricular myocardium—studies by intracellular Ca2+ monitor with indo-1 and by 31P-nuclear magnetic resonance spectroscopy. Jpn Circ J 60:593–603

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Mubagwa K, Lin W, Sipido K, Bosteels S, Flameng W (1997) Monensin-induced reversal of positive force-frequency relationship in cardiac muscle: role of intracellular sodium in rest-dependent potentiation of contraction. J Mol Cell Cardiol 29:977–989. https://doi.org/10.1006/jmcc.1996.0342

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Ng DC, Court NW, dos Remedios CG, Bogoyevitch MA (2003) Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts. Cardiovasc Res 57:333–346

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Oldenburg O, Yang XM, Krieg T, Garlid KD, Cohen MV, Grover GJ, Downey JM (2003) P1075 opens mitochondrial KATP channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts. J Mol Cell Cardiol 35:1035–1042

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Oldenburg O, Qin Q, Krieg T, Yang XM, Philipp S, Critz SD, Cohen MV, Downey JM (2004) Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection. Am J Phys Heart Circ Phys 286:H468–H476. https://doi.org/10.1152/ajpheart.00360.2003

    CAS  Google Scholar 

  42. 42.

    OU J, Komukai K, Kusakari Y, Obata T, Hongo K, Sasaki H, Kurihara S (2005) Alpha1-adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes. Proc Natl Acad Sci U S A 102:9400–9405. https://doi.org/10.1073/pnas.0503569102

    Article  Google Scholar 

  43. 43.

    Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, Downey JM (2000) Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 87:460–466

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Palomeque J, Sapia L, Hajjar RJ, Mattiazzi A, Vila Petroff M (2006) Angiotensin II-induced negative inotropy in rat ventricular myocytes: role of reactive oxygen species and p38 MAPK. Am J Phys Heart Circ Phys 290:H96–106

    CAS  Google Scholar 

  45. 45.

    Palomeque J, Petroff MV, Sapia L, Gende OA, Mundina-Weilenmann C, Mattiazzi A (2007) Multiple alterations in Ca2+ handling determine the negative staircase in a cellular heart failure model. J Card Fail 13:143–154. https://doi.org/10.1016/j.cardfail.2006.11.002

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Palomeque J, Rueda OV, Sapia L, Valverde CA, Salas M, Petroff MV, Mattiazzi A (2009) Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species. Circ Res 105:1204–1212. https://doi.org/10.1161/CIRCRESAHA.109.204172

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Perez NG, Alvarez BV, Camilion de Hurtado MC, Cingolani HE (1995) pHi regulation in myocardium of the spontaneously hypertensive rat. Compensated enhanced activity of the Na+-H+ exchanger. Circ Res 77:1192–1200

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Sabri A, Byron KL, Samarel AM, Bell J, Lucchesi PA (1998) Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes. Circ Res 82:1053–1062

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Satoh H, Blatter LA, Bers DM (1997) Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes. Am J Phys 272:H657–H668

    CAS  Google Scholar 

  50. 50.

    Sepulveda M, Gonano LA, Back TG, Chen SR, Vila Petroff M (2013) Role of CaMKII and ROS in rapid pacing-induced apoptosis. J Mol Cell Cardiol 63:135–145. https://doi.org/10.1016/j.yjmcc.2013.07.013

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Snabaitis AK, Hearse DJ, Avkiran M (2002) Regulation of sarcolemmal Na+/H+ exchange by hydrogen peroxide in adult rat ventricular myocytes. Cardiovasc Res 53:470–480

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Sossalla S, Fluschnik N, Schotola H, Ort KR, Neef S, Schulte T, Wittkopper K, Renner A, Schmitto JD, Gummert J, El-Armouche A, Hasenfuss G, Maier LS (2010) Inhibition of elevated Ca2+/calmodulin-dependent protein kinase II improves contractility in human failing myocardium. Circ Res 107:1150–1161. https://doi.org/10.1161/CIRCRESAHA.110.220418

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Spurgeon HA, duBell WH, Stern MD, Sollott SJ, Ziman BD, Silverman HS, Capogrossi MC, Talo A, Lakatta EG (1992) Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol 447:83–102

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Tsutsui H, Kinugawa S, Matsushima S (2011) Oxidative stress and heart failure. Am J Phys Heart Circ Phys 301:H2181–H2190. https://doi.org/10.1152/ajpheart.00554.2011

    CAS  Google Scholar 

  55. 55.

    Wojtovich AP, Smith CO, Haynes CM, Nehrke KW, Brookes PS (2013) Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel. Biochim Biophys Acta 1827:598–611. https://doi.org/10.1016/j.bbabio.2012.12.007

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Yao Z, Tong J, Tan X, Li C, Shao Z, Kim WC, vanden Hoek TL, Becker LB, Head CA, Schumacker PT (1999) Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes. Am J Phys 277:H2504–H2509

    CAS  Google Scholar 

  57. 57.

    Yeves AM, Caldiz CI, Aiello EA, Villa-Abrille MC, Ennis IL (2015) Reactive oxygen species partially mediate high dose angiotensin II-induced positive inotropic effect in cat ventricular myocytes. Cardiovasc Pathol 24:236–240. https://doi.org/10.1016/j.carpath.2015.01.002

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Yu Y, Zhang L, Yu ZB (2013) Depressed cardiac output at higher pacing rate in isolated working heart of rat. Zhongguo Ying Yong Sheng Li Xue Za Zhi 29:106–109

    PubMed  Google Scholar 

  59. 59.

    Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ (2000) Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med 192:1001–1014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Correspondence to Ernesto A. Aiello or Verónica C. De Giusti.

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All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of La Plata University.

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Espejo, M.S., Aiello, I., Sepúlveda, M. et al. The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase. Pflugers Arch - Eur J Physiol 469, 1663–1673 (2017). https://doi.org/10.1007/s00424-017-2058-z

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Keywords

  • Rat ventricular myocytes
  • Reactive oxygen species
  • p38 MAP kinase
  • Negative staircase
  • Contractility