Molecular adaptation to calsequestrin 2 (CASQ2) point mutations leading to catecholaminergic polymorphic ventricular tachycardia (CPVT): comparative analysis of R33Q and D307H mutants

Abstract

Homozygous calsequestrin 2 (CASQ2) point mutations leads to catecholaminergic polymorphic ventricular tachycardia: a common pathogenetic feature appears to be the drastic reduction of mutant CASQ2 in spite of normal transcription. Comparative biochemical analysis of R33Q and D307H knock in mutant mice identifies different pathogenetic mechanisms for CASQ2 degradation and different molecular adaptive mechanisms. In particular, each CASQ2 point mutation evokes specific adaptive cellular and molecular processes in each of the four adaptive pathways investigated. Thus, similar clinical phenotypes and identical cellular mechanism for cardiac arrhythmia might imply different molecular adaptive mechanisms.

Keywords: CASQ2 mutations; Cathecolaminergic polymorphic ventricular tachycardia; Degradative pathways; Small heat shock proteins.

Fig. 1

Characterization of D307H knock-in hearts. Relative protein expression of CASQ2, GRP78, GRP94, CRT, Bcl-2, STIM1, Orai1, TRPC3 and actin detected by Western Blot analysis in WT and D307H KI hearts. Average percentages for KI mice are given as mean ± SE (n = 4) and are compared to WT expressed as 100%±SE. *p < 0.05, **p < 0.01, ***p < 0.005, Student’ t-test. Blots are representative images of each group and each experiment was repeated at least thrice

Fig. 2

Expression of degradative pathway markers. a Relative protein expression of CNX, Derlin-1, Herp, HRD1, Beclin-1 and actin detected by Western Blot analysis in WT, R33Q KI and D307H KI hearts. b Average percentages are given as mean ± SE (n = 04) and they are compared to WT expressed as 100% ± SE. Asterisks (*) indicate a p < 0.05 as determined by one-way ANOVA Bonferroni’s test. Blots are representative images of each group and each experiment was repeated at least thrice. c Total mono- and poly-ubiquitinated conjugated proteins in WT, R33Q KI and D307H KI hearts detected by Western Blot and on the right, the densitometric profile of the same signal intensities (arbitrary OD) obtained through Scion Image software

Fig. 3

Expresssion of small heat shock proteins. a Relative protein expression of Hsp27, αB-crystallin, Hsp20 and actin detected by Western Blot analysis in WT, R33Q KI, D307H KI and KO hearts. On the right, average percentages of R33Q CASQ2, D307H CASQ2 and KO are given as mean ± SE, n = 4, and are compared to WT expressed as 100% ± SE. b Relative protein expression of phosphorylated forms of Hsp27 (phosphoSer82), αB-crystallin (phosphoSer59 and phosphoSer45), Hsp20 (phosphoSer16) and actin in WT, R33Q KI, D307H KI and KO hearts. On the right, average percentages of R33Q CASQ2, D307H CASQ2 and KO are given as mean ± SE (n = 4) and are compared to WT expressed as 100% ± SE. Asterisks (*) indicate a p < 0.05 as determined by one-way ANOVA Bonferroni’s test. Blots are representative images of each group and each experiment was repeated at least thrice