Marie-Louise Ward

Type 2 diabetes (T2D) is a global epidemic, with heart failure being the leading cause of premature death. Mitochondrial dysfunction, characterized by impaired energy metabolism, weakened energy transport system and increased oxidative stress, has been proposed as a key contributor to the impairment of contractile function in T2D hearts. However, direct evidence from human T2D hearts remains limited. We assessed the mitochondrial function of right atrial tissues obtained from consenting patients undergoing coronary bypass surgery, comparing T2D and non‐diabetic groups. We used high‐resolution respirometry and fluorometry techniques to assess mitochondrial O ₂ flux and H ₂ O ₂ production in permeabilized cardiac fibres in simulated physiological conditions supported by ATP substrate. Our findings showed that O ₂ flux during oxidative phosphorylation and ATP‐stimulated respiration states was similar between groups. T2D fibres exhibited a lower H ₂ O ₂ production rate during the leak state, both per tissue mass and per O ₂ consumed, but no group difference was observed in the oxidative phosphorylation respiratory state. These findings suggest that T2D, in the context of other comorbidities, such as coronary artery disease and obesity, might not contribute significantly to mitochondrial dysfunction in the human heart.

Diabetes impairs myocardial function. This study investigates tissue composition and contractile function of isolated atrial tissue from type 2 diabetic patients prior to heart failure. Multicellular trabeculae were dissected from freshly obtained right atrial appendage samples from consenting patients undergoing heart surgery. Trabeculae were mounted in a stress transducer at optimal length and electrically stimulated to contract. The steady‐state force produced in response to stimulation at physiological frequencies was recorded at 37°C. Myocardial composition of trabeculae from the same patient samples was examined by immunolabeling of contractile proteins, extracellular collagens (types I and III), and fibroblasts. Relative to nondiabetic, diabetic trabeculae had increased diastolic (p = 0.01) and decreased active stress (p = 0.02), with no difference in the time course of contraction and relaxation. Immunohistological findings showed that diabetic trabeculae had reduced myofilament content (p = 0.02), whereas the relative abundance of collagens type I and type III, cardiomyocytes, and fibroblasts was comparable to nondiabetic trabeculae. However, diabetic trabeculae had a significant reduction in the collagen I/III ratio (p = 0.04) with differences in fibroblast morphology. Our study demonstrated that the contractile function at this stage of diabetic heart disease was associated with small changes in myocardial composition in the right atrium.

Type 2 diabetes is associated with a range of adverse health outcomes, including metabolic dysfunction and increased risk of heart failure. Although the interactions between diabetes and heart disease are complex and incompletely understood, they can be more effectively investigated using multiscale, biophysically based models of the underlying physiological processes. In this study, experimental data from non‐diabetic and diabetic human atrial muscles were used to develop metabolite‐sensitive cross‐bridge models representing each group. The parameterisation of these cross‐bridge models revealed that reduced muscle stress development and a leftward shift of the complex modulus measured in the diabetic muscles could be attributed to reduced cross‐bridge stiffness and slower cross‐bridge detachment rates, respectively. These cross‐bridge models were also integrated into muscle models to investigate the effects of diabetic cross‐bridge function, Ca ²⁺ handling and altered metabolite availability on isometric and physiological work‐loop contractions. The diabetic model produced isometric twitches with lower amplitude and prolonged duration and, in response to lowered ATP concentration, the diastolic stress increased notably. In work‐loop simulations, the diabetic model exhibited slower shortening, reduced work output and lower power of shortening. However, it was also more efficient and had a less pronounced negative response to increases in P _i concentration. These simulations demonstrate that while experimentally measured differences in diabetic cardiac tissues can lead to impaired function during physiological contractions, they may also offer compensatory advantages. The insights of this study offer clear mechanisms of mechanoenergetic dysfunction in diabetic heart muscle, identifying potential therapeutic targets to improve cardiac outcomes for individuals with diabetes. image Key points: We used multiscale mathematical models to investigate the interaction between mechanical and energetic systems in diabetic cardiomyopathy. Using previously collected data from human atrial tissues, we developed models of non‐diabetic and diabetic cross‐bridge function. The models confirmed that the mechanisms underlying slower cycling and lower force production in the diabetic tissues were slower cross‐bridge detachment rates and lower cross‐bridge stiffness. Integrating into muscle models and predicting the behaviour under different types of contraction revealed that these differences lead to longer force twitches with lower amplitude, less work done, slower shortening and lower power generation in the diabetic muscles. This modelling study presents novel human atrial cross‐bridge models and offers clear mechanisms of mechanical and energetic dysfunction in diabetic heart muscle with potential pathways for treatment.

Pulmonary artery hypertension (PAH) is characterised by increased pulmonary vascular resistance (PVR) resulting in elevated pressure in the pulmonary artery supplying the pulmonary circulation. Disease of the right ventricle (RV) often manifests as a result of PAH placing excessive pressure on the right side of the heart. Although a relatively rare disease in humans, the impact of sustained PAH is severe, with poor outcomes even in treated individuals. As PAH develops, the blood flow is restricted through the pulmonary arteries and the right ventricle hypertrophies due to the increased strain of pumping blood through the pulmonary circulation. With time, RV hypertrophy progresses to right heart failure, impacting the supply of blood to the left ventricle and systemic circulation. Although right heart failure can currently be treated, it cannot be cured. There is therefore a need for more research into the physiological changes that cause the heart to fail under pressure overload. This review aims to evaluate the monocrotaline (MCT) rat model of PAH as a means of studying the cellular mechanisms associated with the development of RV hypertrophy and right heart failure.

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca²⁺ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca²⁺ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca²⁺ ([Ca²⁺]_mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca²⁺]_cyto transients, increased Ca²⁺ store content, and faster trans-sarcolemmal Ca²⁺ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca²⁺]_mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per μm²) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca²⁺]_mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

The aim of this study was to investigate cardiomyocyte Ca²⁺ handling and contractile function in freshly excised human atrial tissue from diabetic and non‐diabetic patients undergoing routine surgery. Multicellular trabeculae (283 ± 20 μm in diameter) were dissected from the endocardial surface of freshly obtained right atrial appendage samples from consenting surgical patients. Trabeculae were mounted in a force transducer at optimal length, electrically stimulated to contract, and loaded with fura‐2/AM for intracellular Ca²⁺ measurements. The response to stimulation frequencies encompassing the physiological range was recorded at 37°C. Myofilament Ca²⁺ sensitivity was assessed from phase plots and high potassium contractures of force against [Ca²⁺]_i. Trabeculae from diabetic patients (n = 12) had increased diastolic (resting) [Ca²⁺]_i (p = 0.03) and reduced Ca²⁺ transient amplitude (p = 0.04) when compared to non‐diabetic patients (n = 11), with no difference in the Ca²⁺ transient time course. Diastolic stress was increased (p = 0.008) in trabeculae from diabetic patients, and peak developed stress decreased (p ≤ 0.001), which were not accounted for by reduction in the cardiomyocyte, or contractile protein, content of trabeculae. Trabeculae from diabetic patients also displayed diminished myofilament Ca²⁺ sensitivity (p = 0.018) compared to non‐diabetic patients. Our data provides evidence of impaired calcium handling during excitation‐contraction coupling with resulting contractile dysfunction in atrial tissue from patients with type 2 diabetes in comparison to the non‐diabetic. This highlights the importance of targeting cardiomyocyte Ca²⁺ homeostasis in developing more effective treatment options for diabetic heart disease in the future.

Background: Cardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca²⁺ fluxes drive repeated cycles of contraction and relaxation. Ca²⁺ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca²⁺ changes. However, visualizing mitochondrial Ca²⁺ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca²⁺ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes.Methods: Healthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca²⁺ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and β-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca²⁺ Tyrodes containing 1 μM isoproterenol and 150 μM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca²⁺]cyto) and mitochondrial calcium ([Ca²⁺]_mito) transients were made.Results: Myocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to β-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca²⁺ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001).Conclusion: Myocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca²⁺ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca²⁺]_cyto and [Ca²⁺]_mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca²⁺]_mito transients in intact myocytes.

Acute ventricular dilation can evoke mechanically-induced arrhythmias, our study investigated whether Purkinje fibres (PFs) may play a role. Changes in left ventricular (LV) pressure and pseudo-ECGs were measured in isolated, Langendorff-perfused, male Wistar rat hearts in sinus rhythm. The LV endocardial surface was irrigated with experimental agents, via an indwelling catheter. Mechanically-induced arrhythmias were triggered by LV lumen inflation (100μl in 2s) via an indwelling balloon. Arrhythmias occurred as the LV volume was increased and spontaneously ceased within 20s of the onset of LV inflation. Arrhythmias were indexed as an increase in the standard deviation of all R-R intervals (SDRR), the number of ectopic activations and the period of these activations. Following 10s LV endocardial irrigation with Lugol’s solution (IK/I ₂ ) to chemically ablate surface PFs or with 0 Na ⁺ Tyrode, there was a statistically significant attenuation of mechanically-induced arrhythmias. Lugol’s reduced the mechanically-induced increase in SDRR (Tyrode pre-stretch 3.5±1.7ms to 113.8±15.1ms during stretch vs Lugol pre-stretch 3.3±0.5ms to 39.9±14.5ms during stretch n=8, P < 0.05). There was also a reduction in the number (21.2±2.0 to 1.5±0.7, Pin situ focal mechanical stimulation of individual PFs, caused ectopic activations, in each of 4 sheep LV preparations (0.2±0.1 ectopics in 10s pre-mechanical stimulation vs 2.1±0.2 ectopics in 10s during mechanical stimulation, P<0.001). We interpret our observations in rat and sheep hearts as evidence for a role of PFs in the generation of some mechanically-induced arrhythmia.

Aim: Altered organization of the transverse‐tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t‐tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation‐contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t‐tubular network has a direct effect on cardiac mechano‐energetics. Our aim was to understand the fundamental link between t‐tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event.Methods: We exploited the “formamide‐detubulation” technique to acutely disrupt the t‐tubular network in rat left‐ventricular trabeculae. We assessed the energy utilization by cellular Ca²⁺ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured.Results: Following structural disorganization from detubulation, muscle heat output associated with Ca²⁺ cycling was reduced, indicating impaired intracellular Ca²⁺ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force‐length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced.Conclusions: These results reveal the direct energetic consequences of disrupted t‐tubular network, linking the energy disturbance and the t‐tubular remodelling typically observed in heart failure.

Ventricular muscle has a biphasic response to stretch. There is an immediate increase in force that coincides with the stretch which is followed by a second phase that takes several minutes for force to develop to a new steady state. The initial increase in force is due to changes in myofilament properties, whereas the second, slower component of the stretch response (known as the “slow force response” or SFR) is accompanied by a steady increase in Ca²⁺ transient amplitude. Evidence shows stretch-dependent Ca²⁺ influx during the SFR occurs through some mechanism that is continuously active for several minutes following stretch. Many of the candidate ion channels are located primarily in the t-tubules, which are consequently lost in heart disease. Our aim, therefore, was to investigate the impact of t-tubule loss on the SFR in non-failing cardiac trabeculae in which expression of the different Ca²⁺ handling proteins was not altered by any disease process. For comparison, we also investigated the effect of formamide detubulation of trabeculae on β-adrenergic activation (1 μM isoproterenol), since this is another key regulator of cardiac force. Measurement of intracellular calcium ([Ca²⁺]_i) and isometric stress were made in RV trabeculae from rat hearts before, during and after formamide treatment (1.5 M for 5 min), which on washout seals the surface sarcolemmal t-tubule openings. Results showed detubulation slowed the time course of Ca²⁺ transients and twitch force, with time-to-peak, maximum rate-of-rise, and relaxation prolonged in trabeculae at optimal length (L_o). Formamide treatment also prevented development of the SFR following a step change in length from 90 to 100% L_o, and blunted the response to β-adrenergic activation (1 μM isoproterenol).