Adrian C. Grimes

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.

The anatomical components of the cardiac outflow tract of the gray bichir, Polypterus senegalus: their evolutionary significance.

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals.

LRRC10 is required to maintain cardiac function in response to pressure overload

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10(-/-)) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10(-/-) mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10(-/-) mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10(-/-) cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His(150) of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis.

Increased Postnatal Cardiac Hyperplasia Precedes Cardiomyocyte Hypertrophy in a Model of Hypertrophic Cardiomyopathy

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C (MYBPC3) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C−/−) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C−/− phenotype before and after overt hypertrophy. cMyBP-C−/− hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C−/− HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C−/− mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced.

Transcriptome Analysis of Cardiac Hypertrophic Growth in MYBPC3-Null Mice Suggests Early Responders in Hypertrophic Remodeling

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C (MYBPC3) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C-/-) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC-/- mice (n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth (n = 61), physiologic growth (n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C-/- hearts (n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C-/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2, and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C-/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C-/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2, as well as Zbtb16, along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

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}