Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy

Abstract

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 (PLN), calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM compared to controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (p<0.001). The CaMKII target, pT17-PLN, was 5.5-fold increased only in sarcomere-mutation HCM (p=0.01), as was auto-phosphorylated CaMKII (p<0.01) suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating lack of calcineurin activation. Further, MEF2 and NFAT 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 SERCA2/PLN protein ratio (45% reduced, p=0.03).45Ca SERCA uptake assay showed reduced uptake velocity in HCM regardless of genotype (p=0.01). The cardiac ryanodine receptor (RyR2) 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 RyR2. Conclusions Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Post-translational activation of the CaMKII pathway is specific to sarcomere mutation-HCM, while SERCA2 abundance and SR Ca uptake are depressed in both sarcomere mutation-positive and negative HCM.

Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy

“…In addition, we performed prespecified pathway analysis for calcium-handling genes, NMD genes, and hypertrophy-associated genes. Further supporting an absence of calcium mishandling in the early developmental stage, we found no significantly dysregulated calcium-handling genes in either heterozygous or homozygous models ( Figure 5C), including ATPA2A (Serca2) and CAMK2D, both of which are dysregulated in HCM patient cardiac tissue and in mouse models of HCM (28,30). We also found no significant differential expression of NMD genes in MYBPC3 mutation lines, including in frameshift mutation lines (in which NMD degrades mutant mRNA transcripts) versus promoter deletion or start site mutation lines (in which mRNA loss of function occurs without NMD).…”

“…Contractile dysfunction in homozygous MYBPC3-mutant iPSCMs is independent of calcium dynamics. Calcium mishandling has been thought to be a key feature of HCM (27), and we previously demonstrated calcium handling abnormalities specific to sarcomere mutation HCM using patient cardiac tissue samples, including samples with MYBPC3 mutations (28). Intracellular calcium measurements (with fura-2 indicator dye) in heterozygous and homozygous frameshift mutation lines demonstrated normal diastolic calcium level, normal peak calcium levels, and normal calcium transient kinetics (Supplemental Figure 7).…”

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models

“…To identify some of the potential mechanisms underlying the observed Ca 2+ handling differences, fast‐frozen LV myocardial samples from WT, R92Q, and E163R animals were processed to obtain total proteins, which were used for Western blot studies aimed at identifying changes in CaMKII signaling and other molecular markers of cardiac remodelling, as previously described in human myocardium (Figure A) . Notably, CaMKII autophosphorylation, a marker of CaMKII activation, was increased in hearts from both R92Q and E163R compared with WT mice.…”

Calcium‐calmodulin‐dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance

“…Binding assays were carried out following a modified version of a protocol previously described (Helms et al . ). Binding mixtures were prepared containing 50 μg of protein from cell lysates, 0.2 m KCl, 20 m m Na‐Hepes (pH 7.4), 6.5 n m [ 3 H]ryanodine (Perkin Elmer, Boston, MA, USA) and enough CaCl 2 to set free [Ca 2+ ] between 100 n m and 100 μ m .…”

“…Stable, inducible HEK293 cells expressing mouse RyR2 (Helms et al . ) were grown in low glucose Dulbecco's modified Eagle medium (100 mg dl −1 ; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U ml −1 penicillin and 100 μg ml −1 streptomycin on 100‐mm tissue culture dishes. RyR2 expression was induced by adding 1 μg ml −1 tetracycline to the culture medium.…”