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

PLOS ONE, Dec 2019

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.

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

December Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model Manami Kaneko 0 1 2 Kentarou Hashikami 0 2 Satoshi Yamamoto 0 2 Hirokazu Matsumoto 0 2 Tomoyuki Nishimoto 0 1 2 0 Competing Interests: All authors are employees of Takeda Pharmaceutical Company Limit. All 1 Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited , Fujisawa, Kanagawa , Japan , 2 Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited , Fujisawa, Kanagawa , Japan 2 Editor: Junichi Sadoshima, Rutgers New Jersey Medical School , UNITED STATES 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)/CRISPRassociated (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. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was financially supported by Takeda Pharmaceutical Company Limited. The authors as employees of Takeda played their roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Heart failure (HF) is the complicated clinical syndrome characterized by progressive cardiac remodeling and dysfunction. Despite the advances in device therapy [ 1, 2 ] and pharmacological therapy such as angiotensin II receptor blocker [3], angiotensin converting enzyme inhibitors [ 4 ], and β-blockers [ 5 ], HF remains a major cause of morbidity in the world [ 6, 7 ]. Additionally, prevalence of HF is growing due to raise of aging population in the developed countries, therefore more novel and effective treatments for HF is required. Research about the mechanism of HF over the year revealed that one of the most consistent cellular features in HF patients is an impaired Ca2+ homeostasis with alterations in the amplitude and kinetics of Ca2+ transients [8±10]. Ca2+ has critical roles as a second messenger in several signaling pathways in the heart, and this abnormal Ca2+ handling in HF involves contractile dysfunction, remodeling, abnormal electrical activity, reduction of ATP production, and apoptosis [11±13]. Sarco/endoplasmic reticulum calcium ATPase 2a, (SERCA2a) expressed in sarcoplasmic reticulum (SR) and its inhibitory protein called phospholamban (PLN) are major proteins regulating Ca2+ handling. Reductions of SERCA2a protein expression and activity, and an enhancement of inhibitory effect of PLN are associated with impaired SR Ca2+ uptake in cardiomyocytes of HF, subsequently decrease Ca2+ release from SR to cytosol via ryanodine receptor, which induces the reduction of both systolic and diastolic function and, eventually cardiac death [14±16]. Therefore, SERCA2a and PLN are expected as a target for novel therapy of HF for a few decades, indeed, it was reported that the normalization of SERCA2a function and the PLN inhibition increased contractility with correction of Ca2+ homeostasis in a large number of studies in isolated cardiomyocyte and animal models of HF [17±19]. However, positive inotropic agents, such as β-adrenoceptor agonists and PDE III inhibitors, which increase cardiac contractility with increasing cAMP levels and stimulating Ca2+ cycling, caused worsening mortality in HF patients as a result of increased energetic consumption and abnormal electrical activity [ 20, 21 ]. Although similar concerns are raised regarding SERCA2a activation or PLN inhibition therapy which causes enhancement of Ca2+ cycling and contractility, there are few reports about survival in preclinical study. T (...truncated)


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Manami Kaneko, Kentarou Hashikami, Satoshi Yamamoto, Hirokazu Matsumoto, Tomoyuki Nishimoto. Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model, PLOS ONE, 2016, Volume 11, Issue 12, DOI: 10.1371/journal.pone.0168486