Novel Carvedilol Analogues That Suppress Store-Overload-Induced Ca²⁺ Release

Carvedilol is a uniquely effective drug for the treatment of cardiac arrhythmias in patients with heart failure. This activity is in part because of its ability to inhibit store-overload-induced calcium release (SOICR) through the RyR2 channel. We describe the synthesis, characterization, and bioassay of ca. 100 compounds based on the carvedilol motif to identify features that correlate with and optimize SOICR inhibition. A single-cell bioassay was employed on the basis of the RyR2-R4496C mutant HEK-293 cell line in which calcium release from the endoplasmic reticulum through the defective channel was measured. IC₅₀ values for SOICR inhibition were thus obtained. The compounds investigated contained modifications to the three principal subunits of carvedilol, including the carbazole and catechol moieties, as well as the linker chain containing the β-amino alcohol functionality. The SAR results indicate that significant alterations are tolerated in each of the three subunits

Cardiac-specific, conditional knockout of the WT RyR2 allele in heterozygous RyR2 Ex3-del mutant mice results in early death.

<p>iRyR2^flox/flox (n = 13, black circles) and iRyR2^flox/Ex3-del (n = 11, white circles) mice were injected with tamoxifen (75 mg/kg/day) for 3 consecutive days. The percentage of live mice (survival rate) on day 6–14 post tamoxifen treatment is shown.</p

Deletion of exon-3 in the RyR2 mRNA from heterozygous RyR2 Ex3-del mice.

<p>A fragment of the mouse RyR2 mRNA covering exon-3 was converted to cDNA and amplified using RT-PCR from total RNAs isolated from wild type (WT) and heterozygous RyR2 Ex3-del (Het) mutant mice (A). The RT-PCR products were isolated and sequenced. The sequence of the RyR2 Ex3-del cDNA was shown (B). Note that the exon-2 sequence is directly followed by the exon-4 sequence, i.e. the exon-3 sequence has been deleted.</p

Reduced RyR2 protein expression in heterozygous RyR2 Ex3-del mutant hearts.

<p>(A) Whole heart homogenates were prepared from wild type (WT) (n = 4) and RyR2 Ex3-del^−/− mutant (n = 4) mice (2–3 months) and used for immunoblotting analysis using antibodies against RyR2 or β-actin. (B) The expression of RyR2 in the Ex3-del hearts was significantly reduced (58±3%) as compared to that in WT hearts (*<i>P</i><0.001).</p

Depolarization-induced Ca²⁺ transients in WT and heterozygous RyR2 Ex3-del mutant cardiomyocytes.

<p>Ventricular myocytes isolated from RyR2 WT and Ex3-del<b>^+/</b>⁻ mutant hearts were loaded with Rhod-2-AM and perfused with 2 mM extracellular Ca²⁺ in KRH solution and paced at 3Hz. Ca²⁺ transients were monitored by line-scan confocal Ca²⁺ imaging. Representative images/traces of WT (A) and Ex3-del<b>^+/</b>⁻ mutant (B) cardiomyocytes, and average data of the amplitude (C), time to peak (D), and time to 50% decay (E) of Ca²⁺ transients in WT and Ex3-del<b>^+/</b>⁻ mutant cells are shown. Data shown are mean ± SEM from 35 WT and 58 mutant cells (**P<0.001; *P<0.05).</p

Cardiac specific, conditional knockout of the WT RyR2 allele results in markedly reduced RyR2 expression.

<p>Whole heart homogenates were prepared from iRyR2^flox/flox (A) and iRyR2^flox/Ex3-del (B) mice before and after tamoxifen treatment, and used for immunoblotting analysis using antibodies against RyR2 or β-actin. Note that the expression of RyR2 in iRyR2^flox/flox hearts (n = 6) or iRyR2^flox/Ex3-del hearts (n = 6) was significantly reduced 12 days post tamoxifen treatment (*<i>P</i><0.001).</p

Heterozygous RyR2 Ex3-del mutant mice with cardiac specific, conditional KO of the WT RyR2 allele exhibit bradycardia, but no CPVT.

<p>iRyR2^flox/flox (n = 11) and iRyR2^flox/Ex3-del (n = 8) mice were treated with tamoxifen. ECG recording was performed 12 days post tamoxifen treatment to determine their basal heart rates (before epinephrine/caffeine challenge) (A) and their susceptibility to CPVT (B, C). Representative ECG recordings of the tamoxifen-treated iRyR2^flox/flox mice (B) and the tamoxifen-treated iRyR2^flox/Ex3-del mice (C) before (top panel) and after (bottom panel) the injection of epinephrine (1.6 mg/kg) and caffeine (120 mg/kg). Note that no VTs were detected in either the tamoxifen-treated iRyR2^flox/flox mice (B) or the tamoxifen-treated iRyR2^flox/Ex3-del mice during the 30-min period of ECG recording after the injection of the triggers (*P<0.05).</p