Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy.
BACKGROUND
Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways.


METHODS
We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8).


RESULTS
Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03).45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor.


CONCLUSIONS
Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor. adamhelm@med.umich.edu.
Adam S
AS
Helms
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Francisco J
FJ
Alvarado
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Jaime
J
Yob
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Vi T
VT
Tang
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Francis
F
Pagani
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Mark W
MW
Russell
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Héctor H
HH
Valdivia
From Departments of Internal Medicine (A.S.H., J.Y., V.T.T., H.H.V., S.M.D.), Molecular and Integrative Physiology (F.J.A., H.H.V., S.M.D.), Cardiac Surgery (F.P.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor.
Sharlene M
SM
Day
D020013:Calcium Signaling / Q000235:genetics*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D015536:Down-Regulation; D015870:Gene Expression; D005838:Genotype; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000099163:Myosin Binding Protein C
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002352:Carrier Proteins; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099163:Myosin Binding Protein C
calcium; calcium-calmodulin-dependent protein kinase type 2; cardiomyopathy, hypertrophic; genetics; sarcomeres; sarcoplasmic reticulum calcium-transporting ATPases
Proc Natl Acad Sci U S A. 2010 Oct 19;107(42):18097-102
20923879
Circulation. 2007 Nov 27;116(22):2587-96
18025526
Circ Res. 2002 Mar 22;90(5):594-601
11909824
Circ Res. 2012 Jul 6;111(2):170-9
22647877
Circulation. 2006 Sep 26;114(13):1352-9
16982937
Circulation. 2011 Dec 13;124(24):2761-96
22068435
J Mol Cell Cardiol. 2012 Jun;52(6):1299-307
22465693
Circulation. 2013 Feb 5;127(5):575-84
23271797
Circ Res. 2013 Jan 18;112(2):298-308
23152493
J Clin Invest. 2000 Dec;106(11):1351-9
11104788
Mol Cell Biol. 2005 Aug;25(15):6649-59
16024800
Hum Gene Ther. 2015 May;26(5):293-304
25914929
Eur J Heart Fail. 2006 Mar;8(2):115-21
16214409
Circ Res. 2013 May 24;112(11):1491-505
23508784
Am J Physiol Heart Circ Physiol. 2009 Aug;297(2):H614-26
19502551
Circ Res. 2011 Mar 18;108(6):765-82
21415410
Eur Heart J. 2011 May;32(9):1114-20
21345853
J Biol Chem. 2007 Nov 16;282(46):33752-9
17873280
Circ Res. 2003 May 2;92(8):912-9
12676814
Biochem Biophys Res Commun. 2004 Oct 1;322(4):1178-91
15336966
J Am Coll Cardiol. 2010 Sep 7;56(11):867-74
20688032
Sci China Life Sci. 2015 Jan;58(1):54-8
25480325