Toan Pham

Striated muscles differ markedly in energetic demand, but whether mitochondrial respiration and reactive oxygen species emission differ intrinsically across muscle types remains unclear. This study compared mitochondrial respiration, hydrogen peroxide (H ₂ O ₂ ) emission and antioxidant enzyme expression in heart, diaphragm and hindlimb skeletal muscle from healthy rats. Mitochondrial O ₂ flux and H ₂ O ₂ emission were measured simultaneously using high‐resolution respirometry coupled with fluorometry in tissue homogenates subjected to substrate‐uncoupler‐inhibitor titration protocols. Mitochondrial content was estimated by citrate synthase activity, and antioxidant enzyme expression was measured by immunoblotting. Heart tissue displayed the highest mitochondrial respiration and H ₂ O ₂ emission when expressed per tissue mass, followed by diaphragm and skeletal muscle. However, normalisation by citrate synthase activity eliminated differences in respiratory flux between muscles, indicating that variations in tissue‐level respiration were primarily driven by mitochondrial content. Net H ₂ O ₂ emission was greatest during leak respiration and decreased during oxidative phosphorylation and uncoupled states in all muscle types. Cardiac mitochondria exhibited a higher H ₂ O ₂ /O ₂ ratio during leak respiration and higher coupling‐related ratios than those of diaphragm and skeletal muscle. In association, cardiac muscle showed greater expression of the mitochondrial antioxidant enzymes superoxide dismutase 2 and catalase in a separate cohort. These findings indicate that absolute oxidative metabolism across striated muscles is largely determined by mitochondrial content, whereas respiratory capacity per mitochondrial unit was conserved. Cardiac muscle exhibits elevated leak‐associated reactive oxygen species emission but compensates by enhancing antioxidant defences, highlighting coordination between bioenergetic demand and redox regulation in continuously active muscles.

The three‐element Windkessel model of the systemic circulation comprises terms that represent the resistance of peripheral vessels ( R _p ), compliance of the arteries ( C ) and characteristic impedance ( Z _c ) of the aorta. It is widely used to represent the biophysics of the arterial system, and can be used to model the vascular load that can be experimentally presented to isolated hearts or muscles. Reported values of the three Windkessel parameters span several orders of magnitude. By collecting literature data across species, we found empirical relationships that are naturally preserved among the three Windkessel parameters. We applied these empirical relationships in our experiments when prescribing loads to isolated cardiac muscles, and compared the results with loading protocols that do not preserve these empirical relationships. Thus, in our loading protocols, R _p was varied, whereas C and Z _c were either held constant or varied according to their empirical relationship with R _p . Muscles responded to the prescribed loads and generated force–length work‐loops that were unique to the various loading protocols. The differences in work‐loops were most evident during muscle shortening in terms of the force achieved to commence shortening, the dynamics of the force during shortening and the force reached at the completion of shortening. With these loading protocol‐specific shortening, muscles liberated distinct amounts of shortening heat and performed different amounts of work, resulting in different levels of mechanical efficiency. In conclusion, the three‐element Windkessel parameters have empirical relationships that uniquely define the load and mechanoenergetics of cardiac muscle shortening. image Key points: The heart pumps blood into the aorta against the arterial load imposed by the systemic vasculature. The biophysical properties of the vasculature are characterised by resistance, compliance and impedance. By compiling literature data across multiple species, we identified a relation that is rather ‘universal’ linking resistance and compliance, and another linking resistance and impedance. We conducted experiments on isolated cardiac muscles subjected to contract against loads that either conformed to or departed from the relations. We found that when the loads conformed to the relations, the mechanics, kinetics and energetics of muscle contraction and shortening were distinct from those loads that deviated from the relations. These findings establish a fundamental link between arterial load and cardiac muscle mechanoenergetics, demonstrating that presenting isolated muscles with loading conditions that conform to the empirical relations is essential for understanding the physiological mechanics of cardiac contraction.

Type 2 diabetes mellitus (T2DM) is associated with chronic systemic and cardiac inflammation, contributing to the development of diabetic cardiomyopathy. The mitochondrial‐derived peptide mitochondrial open reading frame of the 12S rRNA type‐C (MOTS‐c) has emerged as a promising regulator of metabolic and inflammatory pathways. In this study, we investigated the effects of MOTS‐c treatment on leucine‐rich repeat and pyrin domain‐containing receptor 3 (NLRP3) inflammasome activity in a high‐fat diet and streptozotocin‐induced T2DM rat model. MOTS‐c treatment significantly reduced fasting blood glucose and circulating C‐reactive protein levels, while selectively modulating plasma inflammatory cytokines, including interleukin (IL)‐10 and IL‐1β. Immunohistochemical analysis revealed reduced levels of NLRP3, apoptosis‐associated speck‐like protein containing a caspase activation and recruitment domain (ASC), and cleaved caspase‐1 in left ventricular tissue following MOTS‐c administration. Correlation analyses linked IL‐18 and IL‐1β with elevated markers, including low‐density lipoprotein and uric acid, suggesting interplay between overall health and inflammasome activity. These results indicate that MOTS‐c modulates both systemic and cardiac inflammation in T2DM, providing a novel therapeutic approach for reducing cardiovascular risk in diabetic patients.

Type 2 diabetes (T2D) greatly alters cardiac fuel handling, yet how mitochondrial function adapts to the diabetic substrate environment remains unclear. This study investigated substrate‐specific cardiac mitochondrial bioenergetics from a T2D rat model induced by a high‐fat diet and low‐dose streptozotocin. High‐resolution respirometry and fluorimetry were used to measure mitochondrial O ₂ flux, ATP flux, reactive oxygen species production rate and mitochondrial membrane potential in cardiac tissue homogenates in two key substrate conditions: carbohydrate and fatty acid. Liquid chromatography–mass spectrometry was used to analyse the abundance of metabolites involved in the Krebs cycle and in various fatty acid metabolic pathways. Carbohydrate‐supported mitochondrial respiration, ATP flux, reactive oxygen species production and mitochondrial membrane potential were preserved in T2D myocardium. In contrast, fatty acid‐supported mitochondrial respiration and ATP flux in oxidative phosphorylation were significantly decreased despite increased myocardial abundance of several fatty acid species, including palmitoleic acid, cis ‐8‐heptadecenoic acid and linoleic acid. Metabolite intermediates of the Krebs cycle were largely unchanged. These findings reveal a substrate‐specific energetic defect in the diabetic heart, in which excess fatty acid supply is not matched by mitochondrial oxidative capacity, leading to metabolic inflexibility and impaired ATP generation. This work provides mechanistic insight into how nutrient overload contributes to mitochondrial inefficiency in T2D and establishes a foundation for future studies targeting lipid–mitochondria interactions.

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.

The left ventricle (LV) is the primary pumping chamber of the heart, generating high systolic pressure to sustain systemic circulation. LV contractile dysfunction is a hallmark of various cardiovascular diseases and is associated with mitochondrial dysfunction, characterised by decreased oxidative phosphorylation (OXPHOS) capacity and increased oxidative stress. While our understanding of cardiac mitochondrial physiology has been gained from studies on LV tissues in animal models or atrial tissues in human studies, findings are often generalised across cardiac regions. Given that fundamental differences in anatomical structure, physiological function and metabolic demands exist between the LV and left atrium (LA), this study aimed to compare mitochondrial bioenergetics between LV and LA tissues from healthy rat hearts. Using high‐resolution respirometry coupled with fluorimetry, we assessed mitochondrial respiration, ATP production and hydrolysis, and reactive oxygen species (ROS) production rates. Protein expression of mitochondrial respiratory complexes and antioxidant enzymes was quantified using western blotting. Our results showed that per tissue mass, LV tissues exhibited greater mitochondrial OXPHOS respiration, ATP production and hydrolysis rates, ROS production rate, and higher protein levels of mitochondrial complexes and antioxidant enzymes, consistent with higher citrate synthase activity as a marker of mitochondrial content. However, when normalised to mitochondrial content, LV tissues exhibited lower OXPHOS respiration and ATP production, expression of mitochondrial complexes and antioxidant proteins compared to LA. This study provides new insights into chamber‐specific differences in mitochondrial function under physiological conditions, suggesting the importance of considering regional mitochondrial profiles in studies of cardiac mitochondrial function in health and disease.

Isolated cardiac trabeculae are small heart muscle tissue preparations, which have been widely used in in vitro studies of mechanics and energetics function of cardiac muscle. Current instruments for such experimentation often (1) involve delicate mounting of the muscle, (2) constrain investigations to one muscle at a time, and thus (3) limit experimental throughput. Here, we present a novel device that allows trabeculae to be secured by a visible‐light photo‐crosslinked hydrogel, manipulated via a robust motor‐driven stainless steel cantilever, and their shortening and force production to be measured and controlled using feedback from real‐time imaging. The device has multiple wells, making it amenable to high‐throughput testing of muscle. We use our robust, accurate image registration techniques to measure cantilever and gel deformation during trabecula contraction and thereby provide a measure of trabecula shortening and force production during twitches. We apply methods to allow the trabecula to contract either isometrically or isotonically. The methods used in this device can be widely applied to the study of the mechanics of cardiac muscle samples in laboratories with available light microscopic systems.

Danicamtiv is a recently developed cardiac‐specific myosin activator that directly increases actomyosin interaction and, thereby, increases force. It is currently studied pre‐clinically and has entered clinical trials. In this study, we provide the first assessment of its effects on cardiac energetics. Ventricular trabeculae from rats were isolated and studied in vitro. Each trabecula was stimulated to produce twitch force over ranges of muscle lengths and afterloads, with muscle heat output simultaneously measured. Each trabecula was superfused with and without danicamtiv (5 µM). In both interventions, muscles were required to undergo both isometric and work‐loop contraction protocols to quantify the components of heat output – that from muscle contraction, that from intracellular Ca²⁺ cycling and that from actomyosin cross‐bridge cycling. Danicamtiv was found to increase twitch force, muscle heat and cross‐bridge heat, with no effect on the energy expenditure associated with intracellular Ca²⁺ cycling. Danicamtiv slowed isometric twitch force kinetics and slowed shortening kinetics. Danicamtiv increased the extent of muscle shortening, increased muscle work output and increased mechanical efficiency, while preserving cross‐bridge efficiency. In conclusion, danicamtiv increases mechanical efficiency, the mechanism of which arises from the greater increase of force and muscle shortening that increase mechanical work than the increase of heat. The relative smaller increase of heat is due to a null effect of danicamtiv on the energy for intracellular Ca²⁺ cycling. Our results provide perspective for the utility of danicamtiv as an inotropic agent not only for mechanical enhancement but also for energetic enhancement in increasing mechanical efficiency. image Key points: Clinical trials are presently exploring the potential of a new myotrope, danicamtiv, to improve cardiac mechanics by increasing muscle force output. A range of active pre‐clinical research is likewise uncovering the biophysical mechanism of how danicamtiv affects cardiac mechanics, increases cardiac force and slows contractile kinetics. This study provides the first energetic assessment of danicamtiv by measuring muscle heat output, and shows that danicamtiv increases cardiac energy efficiency. The mechanism of the increased efficiency resides in the increase of muscle force–length work greater than the increase of muscle heat. This study demonstrates that danicamtiv is a cardiac myotrope that not only enhances mechanics, but also exerts its enhancement effect on energetics.