Hemodynamic parameters.

<p>Cardiac catheterization was performed in CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) dP/dt_max, (B) dP/dt_min, (C) Tau, (D) LVEDP, (E) MBP, (F) HR. Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, ^#P < 0.05 vs. PLN WT/CSQ-Tg mice by Student's <i>t</i>-test.</p

sgRNAs and ssODN targeting <i>Pln</i> coding region.

<p>Targeting sequences of each sgRNA are capitalized and NGG PAM (protospacer-adjacent motif) sequences are underlined. Exons are indicated by closed boxes. The sgRNA targeting sites were designed to sandwich <i>Pln</i> coding region (filled with black). The ssODN is containing homologies of 60 bases on both sides flanking each of the sgRNA targeting sequences.</p

Number of transferred embryos, pups, PLN homozygous KO mice, and PLN wild type (WT) mice in five lots.

<p>Number of transferred embryos, pups, PLN homozygous KO mice, and PLN wild type (WT) mice in five lots.</p

Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model

<div><p>Sarcoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) and its inhibitory protein called phospholamban (PLN) are pivotal for Ca²⁺ handling in cardiomyocyte and are known that their expression level and activity were changed in the heart failure patients. To examine whether PLN inhibition can improve survival rate as well as cardiac function in heart failure, we performed PLN ablation in calsequestrin overexpressing (CSQ-Tg) mice, a severe heart failure model, using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system. According this method, generation rate of PLN wild type mice (PLN copy >0.95) and PLN homozygous knockout (KO) mice (PLN copy <0.05) were 39.1% and 10.5%, respectively. While CSQ overexpression causes severe heart failure symptoms and premature death, a significant ameliorating effect on survival rate was observed in PLN homozygous KO/CSQ-Tg mice compared to PLN wild type/CSQ-Tg mice (median survival days are 55 and 50 days, respectively). Measurement of cardiac function with cardiac catheterization at the age of 5 weeks revealed that PLN ablation improved cardiac function in CSQ-Tg mice without affecting heart rate and blood pressure. Furthermore, increases in atrial and lung weight, an index of congestion, were significantly inhibited by PLN ablation. These results suggest that PLN deletion would be a promising approach to improve both mortality and cardiac function in the heart failure.</p></div

mRNA levels in the left ventricle.

<p>Analysis of mRNA levels was performed in the left ventricle of CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) β-MHC, (B) ANF, (C) BNF. Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, ^#P < 0.05 vs. PLN WT/CSQ-Tg mice by Student's <i>t</i>-test.</p

Number of CSQ-Tg and Non-Tg mice in five lots.

<p>Number of CSQ-Tg and Non-Tg mice in five lots.</p

Heart and lung weight.

<p>Heart and lung weight were measured in CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) LV/BW (left ventricular weight/body weight), (B) RV/BW (right ventricular weight/body weight), (C) AW/BW (atria weight/body weight), (D) LW/BW (lung weight/body weight). Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, ^#P < 0.05, ^##P < 0.01 vs. PLN WT/CSQ-Tg mice by Student's <i>t</i>-test.</p

Immunoblots of PLN and SERCA2a, and actin in hearts from mice used for hemodynamic study.

<p>(A) CSQ non-Tg mice, (B) CSQ-Tg mice.</p

PCR analysis of mice obtained from 1st lot microinjection.

<p>(A) Genotyping of PLN KO mice by PCR. PLN wild type (WT) mice showed 1213 bp fragments and PLN KO mice showed 665 bp fragments. (B) Genotyping of CSQ-Tg mice by PCR. Mice which had canine <i>Csq</i> allele shows 407 bp fragments and Non-Tg mice does not show any fragments. M, DNA marker.</p

Design, Synthesis, and Evaluation of the Highly Selective and Potent G‑Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure

A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative <b>5</b> and 1,2,4-triazole derivative <b>24a</b> were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an <i>N</i>-benzylcarboxamide moiety with highly potent activity toward GRK2 and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, and 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the discovery of <b>115h</b> (GRK2 IC₅₀ = 18 nM), which was obtained the cocrystal structure with human GRK2 and an inhibitor of GRK2 that potentiates β-adrenergic receptor (βAR)-mediated cAMP accumulation and prevents internalization of βARs in β2AR-expressing HEK293 cells treated with isoproterenol. Therefore, <b>115h</b> appears to be a novel class of therapeutic for heart failure treatment