Characterising cellular and molecular mechanisms of cardiac diastolic dysfunction
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-06-05.
© 2019 Dr. Antonia Johanna Adriana Raaijmakers
Background: Diastolic dysfunction is an important contributor to many cardiac pathologies including diabetic cardiomyopathy and heart failure with preserved ejection fraction. It is characterised by ventricular stiffness, inadequate filling of the ventricles and elevated ventricular pressure. In addition to extracellular influences, evidence suggests that a cardiomyocyte specific intrinsic stiffness may also be an important contributor to diastolic dysfunction, but the mechanisms are not well understood. This might be partly due to the lack of specific animal models available to study underlying mechanisms, in particular in HFpEF. The aim of this thesis was to evaluate cellular and molecular mechanisms of diastolic dysfunction in a model of type 1 diabetes and in a newly characterised model of HFpEF, the Hypertrophic Heart Rat (HHR). Research questions: Q1. Can measurement of in vitro intact cardiomyocyte stiffness be correlated with in vivo diastolic function to determine whether cellular stiffness contributes to cardiac diastolic dysfunction in pathological settings? (Chapter 2) Q2. What are the subcellular mechanisms that contribute to increased stiffness in a pathological model of diastolic dysfunction? (Chapter 3) Q3. Can the Hypertrophic Heart Rat be used as a novel rodent model of HFpEF and what is the underlying cardiomyocyte pathophysiology driving diastolic dysfunction in HFpEF? (Chapter 4) Methods: Type 1 diabetes was induced in Sprague Dawley rats using a single dosage of Streptozotocin. The Hypertrophic Heart Rat (HHR) was characterised and utilised as a model of HFpEF. Echocardiography was used to assess in vivo heart function in diabetic and HFpEF rats. Surface electrocardiogram recordings were performed to assess in vivo electrical activity in HFpEF rats. Cardiomyocytes were isolated by collagenase dissociation. Under loaded conditions glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1Hz, 2.0mM Ca2+, 37°C) were serially stretched (011.2%, piezomotor). Force development and intracellular Ca2+ transients (Fura-2AM, 5µM) were simultaneously measured (Myostretcher, IonOptix). In the HHR, histological analysis was undertaken to evaluate collagen deposition. Intracellular Ca2+ and contractility was measured in single unloaded cardiomyocytes (4Hz, 2.0mM Ca2+, 37°C). Left ventricular tissue was homogenised and used for Western blot analysis of Ca2+ handling proteins. Results: A1. Validation of in vivo and in vitro methodologies for the measurement of cardiomyocyte and cardiac diastolic function along with confirmation that in vitro cardiomyocyte stiffness directly correlates to in vivo cardiac dysfunction. This verifies the contribution of cellular stiffness to cardiac diastolic dysfunction in the pathological setting. (Chapter 2) A2. Cardiomyocyte stiffness was shown to be an important contributor to diastolic dysfunction in the diabetic heart. The additive contribution of myofilament cooperativity reduction and slowed Ca2+ reuptake were found to be the subcellular mechanisms for the intracellular stiffness. (Chapter 3) A3. A new model of HFpEF was successfully characterised which closely mirrors clinical pathology without surgical or drug intervention. Animals display early mortality, with cardiac diastolic dysfunction, preserved ejection fraction and arrhythmias. The cardiomyocyte pathology was one of hypercontractility and Ca2+ overload, contrasting strongly with what has been reported in systolic failure leading to potential new therapeutic targets for HFpEF treatment. (Chapter 4) Conclusion: This thesis demonstrates that intact cardiomyocyte stiffness contributes directly to cardiac diastolic dysfunction, which was validated in two separate pathological models. Importantly, this is the first evidence that there is an increase in the slope of the end diastolic force length relation in intact diabetic cardiomyocytes indicating increased cellular stiffness. This was linked to changes in Ca2+ reuptake during relaxation and reduced myofilament cooperativity. In addition, a newly characterized model of HFpEF was described, along with cellular and molecular changes that are apparent in this model of diastolic dysfunction, providing new insight and potentially leading to new therapeutic targets to treat HFpEF. Taken together, this thesis advances the mechanistic understanding of the cellular and molecular mechanisms of diastolic dysfunction.
Keywordscardiac; diastolic function; diabetes; heart failure with preserved ejection fraction; hypertrophy; cell isolation; cardiomyocyte force; intact cardiomyocytes; cell stretch; calcium handling; echocardiography; Myostretcher
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- Physiology - Theses