Carl W. Tong

Acceleration of Crossbridge Kinetics by Protein Kinase A Phosphorylation of Cardiac Myosin Binding Protein C Modulates Cardiac Function

Normal cardiac function requires dynamic modulation of contraction. β1-Adrenergic–induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non–PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, krel (s-1), and delayed force development, kdf (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline krel and kdf similar to WT myocardium, but, unlike WT, krel and kdf were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.

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.nnObjective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy.nnMethods 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.nnConclusions: 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.nn# Novelty and Significance {#article-title-47}

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

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.nnObjective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy.nnMethods 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.nnConclusions: 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.nn# Novelty and Significance {#article-title-47}

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or β1-Adrenergic Stimulation

Background—Mammalian hearts exhibit positive inotropic responses to &bgr;-adrenergic stimulation as a consequence of protein kinase A–mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. Methods and Results—The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca2+]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or &bgr;1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca2+]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKII&dgr;, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. Conclusions—Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with &bgr;-adrenergic stimulation or increased pacing because of protein kinase A and CaMKII&dgr; phosphorylations of cMyB-C.

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.nnObjective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy.nnMethods 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.nnConclusions: 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.nn# 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.nnObjective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy.nnMethods 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.nnConclusions: 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.nn# Novelty and Significance {#article-title-47}