dATP elevation induces myocardial metabolic remodeling to support improved cardiac function.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs.

Impact of etiology on force and kinetics of left ventricular end-stage failing human myocardium.

BACKGROUND: Heart failure (HF) is associated with highly significant morbidity, mortality, and health care costs. Despite the significant advances in therapies and prevention, HF remains associated with poor clinical outcomes. Understanding the contractile force and kinetic changes at the level of cardiac muscle during end-stage HF in consideration of underlying etiology would be beneficial in developing targeted therapies that can help improve cardiac performance. OBJECTIVE: Investigate the impact of the primary etiology of HF (ischemic or non-ischemic) on left ventricular (LV) human myocardium force and kinetics of contraction and relaxation under near-physiological conditions. METHODS AND RESULTS: Contractile and kinetic parameters were assessed in LV intact trabeculae isolated from control non-failing (NF; n = 58) and end-stage failing ischemic (FI; n = 16) and non-ischemic (FNI; n = 38) human myocardium under baseline conditions, length-dependent activation, frequency-dependent activation, and response to the ß-adrenergic stimulation. At baseline, there were no significant differences in contractile force between the three groups; however, kinetics were impaired in failing myocardium with significant slowing down of relaxation kinetics in FNI compared to NF myocardium. Length-dependent activation was preserved and virtually identical in all groups. Frequency-dependent activation was clearly seen in NF myocardium (positive force frequency relationship [FFR]), while significantly impaired in both FI and FNI myocardium (negative FFR). Likewise, ß-adrenergic regulation of contraction was significantly impaired in both HF groups. CONCLUSIONS: End-stage failing myocardium exhibited impaired kinetics under baseline conditions as well as with the three contractile regulatory mechanisms. The pattern of these kinetic impairments in relation to NF myocardium was mainly impacted by etiology with a marked slowing down of kinetics in FNI myocardium. These findings suggest that not only force development, but also kinetics should be considered as a therapeutic target for improving cardiac performance and thus treatment of HF.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium.

BACKGROUND: In patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. METHODS AND RESULTS: The human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the ß-adrenergic response to isoproterenol was depressed in both pathologies. CONCLUSIONS: Force development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

Low levels of Survival Motor Neuron protein are sufficient for normal muscle function in the SMNΔ7 mouse model of SMA.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons. SMA is caused by deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The loss of SMN1 results in reduced levels of the SMN protein. SMN levels appear to be particularly important in motor neurons; however SMN levels above that produced by two copies of SMN2 have been suggested to be important in muscle. Studying the spatial requirement of SMN is important in both understanding how SMN deficiency causes SMA and in the development of effective therapies. Using Myf5-Cre, a muscle-specific Cre driver, and the Cre-loxP recombination system, we deleted mouse Smn in the muscle of mice with SMN2 and SMNΔ7 transgenes in the background, thus providing low level of SMN in the muscle. As a reciprocal experiment, we restored normal levels of SMN in the muscle with low SMN levels in all other tissues. We observed that decreasing SMN in the muscle has no phenotypic effect. This was corroborated by muscle physiology studies with twitch force, tetanic and eccentric contraction all being normal. In addition, electrocardiogram and muscle fiber size distribution were also normal. Replacement of Smn in muscle did not rescue SMA mice. Thus the muscle does not appear to require high levels of SMN above what is produced by two copies of SMN2 (and SMNΔ7).

Prednisolone attenuates improvement of cardiac and skeletal contractile function and histopathology by lisinopril and spironolactone in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited disease that causes striated muscle weakness. Recently, we showed therapeutic effects of the combination of lisinopril (L), an angiotensin converting enzyme (ACE) inhibitor, and spironolactone (S), an aldosterone antagonist, in mice lacking dystrophin and haploinsufficient for utrophin (utrn(+/-);mdx, het mice); both cardiac and skeletal muscle function and histology were improved when these mice were treated early with LS. It was unknown to what extent LS treatment is effective in the most commonly used DMD murine model, the mdx mouse. In addition, current standard-of-care treatment for DMD is limited to corticosteroids. Therefore, potentially useful alternative or additive drugs need to be both compared directly to corticosteroids and tested in presence of corticosteroids. We evaluated the effectiveness of this LS combination in the mdx mouse model both compared with corticosteroid treatment (prednisolone, P) or in combination (LSP). We tested the additional combinatorial treatment containing the angiotensin II receptor blocker losartan (T), which is widely used to halt and treat the developing cardiac dysfunction in DMD patients as an alternative to an ACE inhibitor. Peak myocardial strain rate, assessed by magnetic resonance imaging, showed a negative impact of P, whereas in both diaphragm and extensor digitorum longus (EDL) muscle contractile function was not significantly impaired by P. Histologically, P generally increased cardiac damage, estimated by percentage area infiltrated by IgG as well as by collagen staining. In general, groups that only differed in the presence or absence of P (i.e. mdx vs. P, LS vs. LSP, and TS vs. TSP) demonstrated a significant detrimental impact of P on many assessed parameters, with the most profound impact on cardiac pathology.

The force-temperature relationship in healthy and dystrophic mouse diaphragm; implications for translational study design.

In the field of muscular dystrophy, striated muscle function is often assessed in vitro in dystrophin-deficient mdx mice in order to test the impact of a potential treatment strategy. Although many past studies have assessed diaphragm contractile function at or near room temperature, the diaphragm performs in vivo at 37°C. To improve translation of bench-top results to possible clinical application, we studied temperature-dependence of contractile performance in wild-type (C57BL/10) and mdx muscle strips at temperatures from 25°C to 37°C. Maximal tetanic force in wild-type muscles was higher at 37°C (198 ± 11 vs. 155 ± 9 mN/mm(2) at 25°C), while the difference between wild-type and mdx was extremely similar: wild-type muscles produced 45.9% and 45.1% more force at 25°C and 37°C respectively. At 37°C twitch contraction kinetics and 50% rise time to tetanic plateau were slower in mdx diaphragm. A fatigue/injury protocol indicated 2-fold fatigue/contraction-induced force deficit in mdx muscles. We conclude that assessment of diaphragm muscle strips can be reliably and reproducibly performed at 37°C.

Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice.

BACKGROUND: Nearly universal cardiomyopathy in Duchenne muscular dystrophy (DMD) contributes to heart failure and death. Because DMD patients show myocardial fibrosis well before functional impairment, we postulated that earlier treatment using drugs with antifibrotic effect may be beneficial. METHODS AND RESULTS: Three groups of 10 utrn(+/-);mdx, or "het" mice, deficient for dystrophin and haploinsufficient for utrophin with skeletal myopathy and cardiomyopathy that closely mimics clinical DMD were studied. One het group received spironolactone and lisinopril starting at 8 weeks of life (het-treated-8); a second received the same starting at 4 weeks of life (het-treated-4), and the third het group was untreated. At 20 weeks, all mice had normal ejection fractions though circumferential strain rate was abnormal (-0.21±0.08) in untreated hets. This improved to -0.40±0.07 in het-treated-8 mice (P=0.003) and further improved to -0.56±0.10 in het-treated-4 mice (P=0.014 for het-treated-4 versus het-treated-8). Treated mice showed less cardiomyocyte damage, with a 44% reduction in intracardiomyocyte serum immunoglobulin G localization in het-treated-8 mice (P<0.0001) and a further 53% reduction in het-treated-4 mice (P=0.0003 versus het-treated-8); matrix metalloproteinases were similarly reduced. Cardiac, limb, and diaphragm function by ex vivo muscle testing remained at 80% of normal with early treatment compared to a decline to 40% of normal skeletal muscle function without treatment. CONCLUSIONS: These findings offer clinically available medications with proven antifibrotic effect as a new therapeutic strategy in DMD. Early initiation greatly attenuated myocardial disease and, for the first time with these drugs, improved skeletal myopathy. Thus, early initiation of such agents warrants further clinical evaluation to maintain ambulatory, respiratory, and cardiac function for patients with DMD and related myopathies.