Laura F. Hegge

Neonatal Mouse―Derived Engineered Cardiac Tissue: A Novel Model System for Studying Genetic Heart Disease

Rationale: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. Objective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. Methods and Results: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)–null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C–null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C–null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C−/− ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. Conclusions: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system. # Novelty and Significance {#article-title-47}

Neonatal Mouse–Derived Engineered Cardiac Tissue

Rationale: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. Objective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. Methods and Results: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)–null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C–null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C–null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C−/− ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. Conclusions: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system. # Novelty and Significance {#article-title-47}

Ablation of cardiac myosin–binding protein-C accelerates contractile kinetics in engineered cardiac tissue

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin–binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C–null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca2+ transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C–null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca2+ transient kinetics. Strikingly, the ability of cMyBP-C–null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca2+ transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.

ROSA26 mice carry a modifier of Min-induced mammary and intestinal tumor development.

Abstract. B6.129S7-Gtrosa26 (B6.R26) mice carry a LacZ-neoR insertion on Chromosome (Chr) 6, made by promoter trapping with 129 ES cells. Female C57BL/6J ApcMin/+ (B6Min/+) mice are highly susceptible to intestinal tumors and to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, B6.R26/+Min/+ females develop fewer mammary and intestinal tumors after ENU treatment than do B6 Min/+ mice. B6.R26/+ mice from two independently derived congenic lines show this modifier effect. Each of these congenic lines carries approximately 20 cM of 129-derived DNA flanking the insertion, raising the possibility that the resistance is due to a linked modifier locus. To further map the modifier locus, we have generated several lines of mice carrying different regions of the congenic interval. We have found that resistance to mammary and intestinal tumors in ENU-treated Min/+ mice maps to a minimum 4-cM interval that includes the ROSA26 LacZ-neoR insertion. Therefore, the resistance to tumor development is due to either the ROSA26 insertion or a very tightly linked modifier locus.

The ROSA26 LacZ-neo(R) insertion confers resistance to mammary tumors in Apc(Min/+) mice.

B6.129S7-Gtrosa26 (ROSA26) mice carry a LacZ-neoR insertion on Chromosome (Chr) 6, made by promoter trapping with AB1 129 ES cells. Female C57BL/6J ApcMin/+ (B6 Min/+) mice are very susceptible to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, ENU-treated B6 mice carrying both ApcMin and ROSA26 are resistant to mammary tumor formation. Thus, ROSA26 mice carry a modifier of Min-induced mammary tumor susceptibility. We have previously mapped the modifier to a 4-cM interval of 129-derived DNA that also contains the ROSA26 insertion. Here we report additional evidence for the effect of the ROSA26 insertion on mammary tumor formation. To test the hypothesis that the resistance was due to a linked modifier locus, we utilized two approaches. We have derived and tested two lines of mice that are congenic for 129-derived DNA within the minimal modifier interval and show that they are as susceptible to mammary tumors as are B6 mice. Additionally, we analyzed a backcross population segregating for the insertion and show that mice carrying the insertion are more resistant to mammary tumor development than are mice not carrying the insertion. Thus, the resistance is not due to a 129-derived modifier allele, but must be due to the ROSA26 insertion. In addition, the effect of the ROSA26 insertion can be detected in a backcross population segregating for other mammary modifiers.

Neonatal Mouse–Derived Engineered Cardiac TissueNovelty and Significance

Rationale: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. Objective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. Methods and Results: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)–null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C–null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C–null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C−/− ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. Conclusions: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system. # Novelty and Significance {#article-title-47}

Neonatal Mouse–Derived Engineered Cardiac TissueNovelty and Significance: A Novel Model System for Studying Genetic Heart Disease

Rationale: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. Objective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. Methods and Results: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)–null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C–null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C–null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C−/− ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. Conclusions: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system. # Novelty and Significance {#article-title-47}