Expanding landscape of coronary microvascular disease in co-morbid conditions: Metabolic disease and beyond.

Coronary microvascular disease (CMD) and impaired coronary blood flow control are defects that occur early in the pathogenesis of heart failure in cardiometabolic conditions, prior to the onset of atherosclerosis. In fact, recent studies have shown that CMD is an independent predictor of cardiac morbidity and mortality in patients with obesity and metabolic disease. CMD is comprised of functional, structural, and mechanical impairments that synergize and ultimately reduce coronary blood flow in metabolic disease and in other co-morbid conditions, including transplant, autoimmune disorders, chemotherapy-induced cardiotoxicity, and remote injury-induced CMD. This review summarizes the contemporary state-of-the-field related to CMD in metabolic and these other co-morbid conditions based on mechanistic data derived mostly from preclinical small- and large-animal models in light of available clinical evidence and given the limitations of studying these mechanisms in humans. In addition, we also discuss gaps in current understanding, emerging areas of interest, and opportunities for future investigations in this field.

Machine Learning: a new era for cardiovascular pregnancy physiology and cardio-obstetrics research.

The maternal cardiovascular system undergoes functional and structural adaptations during pregnancy and postpartum to support increased metabolic demands of offspring and placental growth, labor, and delivery, as well as recovery from childbirth. Pregnancy thus poses physiological stress upon the maternal cardiovascular system, and in the absence of an appropriate response it imparts potential risks for cardiovascular complications and adverse outcomes. The proportion of pregnancy-related maternal deaths from cardiovascular events has been steadily increasing, contributing to high rates of maternal mortality. Despite advances in cardiovascular physiology research, there is still no comprehensive understanding of maternal cardiovascular adaptations in healthy pregnancies, and with far less known about pregnancy with complications. Further, current tools for prognosis of cardiovascular complications during pregnancy are limited. Machine learning (ML) offers new and effective tools for investigating mechanisms involved in pregnancy-related cardiovascular complications as well as the development of potential therapies. The main goal of this review is to summarize existing research that uses ML to understand mechanisms of cardiovascular physiology during pregnancy and develop prediction models for clinical application in pregnant patients. We also provide an overview of ML fundamentals and a discussion about platforms that can be used to enhance understanding of cardiovascular adaptations to pregnancy. Finally, we address the interpretability and explainability of ML outcomes, consequences of model bias, and ethics of ML use.

Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.

ΔR4-R23/ΔCT micro-dystrophin (µDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, µDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of µDys, we compared by mass spectrometry the composition of purified dystrophin and µDys protein complexes in the mouse heart. We report that compared to dystrophin, µDys has altered associations with α1- and ß2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of µDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.

Sex differences in vascular aging and impact of GPER deletion.

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and endothelial dysfunction. We hypothesized that sex differences exist in vascular aging processes and would be attenuated by global deletion of the G protein-coupled estrogen receptor. Blood pressure was measured by tail-cuff plethysmography, pulse wave velocity (PWV) and echocardiography were assessed with high-resolution ultrasound, and small vessel reactivity was measured using wire myography in adult (25 wk) and middle-aged (57 wk) male and female mice. Adult female mice displayed lower blood pressure and PWV, but this sex difference was absent in middle-aged mice. Aging significantly increased PWV but not blood pressure in both sexes. Adult female carotids were more distensible than males, but this sex difference was lost during aging. Acetylcholine-induced relaxation was greater in female than male mice at both ages, and only males showed aging-induced changes in cardiac hypertrophy and function. GPER deletion removed the sex difference in PWV and ex vivo stiffness in adult mice. The sex difference in blood pressure was absent in KO mice and was associated with endothelial dysfunction in females. These findings indicate that the impact of aging on arterial stiffening and endothelial function is not the same in male and female mice. Moreover, nongenomic estrogen signaling through GPER impacted vascular phenotype differently in male and female mice. Delineating sex differences in vascular changes during healthy aging is an important first step in improving early detection and sex-specific treatments in our aging population.NEW & NOTEWORTHY Indices of vascular aging were different in male and female mice. Sex differences in pulse wave velocity, blood pressure, and large artery stiffness were abrogated in middle-aged mice, but the female advantage in resistance artery vasodilator function was maintained. GPER deletion abrogated these sex differences and significantly reduced endothelial function in adult female mice. Additional studies are needed to characterize sex differences in vascular aging to personalize early detection and treatment for vascular diseases.

Chronic high-rate pacing induces heart failure with preserved ejection fraction-like phenotype in Ossabaw swine.

The lack of pre-clinical large animal models of heart failure with preserved ejection fraction (HFpEF) remains a growing, yet unmet obstacle to improving understanding of this complex condition. We examined whether chronic cardiometabolic stress in Ossabaw swine, which possess a genetic propensity for obesity and cardiovascular complications, produces an HFpEF-like phenotype. Swine were fed standard chow (lean; n = 13) or an excess calorie, high-fat, high-fructose diet (obese; n = 16) for ~ 18 weeks with lean (n = 5) and obese (n = 8) swine subjected to right ventricular pacing (180 beats/min for ~ 4 weeks) to induce heart failure (HF). Baseline blood pressure, heart rate, LV end-diastolic volume, and ejection fraction were similar between groups. High-rate pacing increased LV end-diastolic pressure from ~ 11 ± 1 mmHg in lean and obese swine to ~ 26 ± 2 mmHg in lean HF and obese HF swine. Regression analyses revealed an upward shift in LV diastolic pressure vs. diastolic volume in paced swine that was associated with an ~ twofold increase in myocardial fibrosis and an ~ 50% reduction in myocardial capillary density. Hemodynamic responses to graded hemorrhage revealed an ~ 40% decrease in the chronotropic response to reductions in blood pressure in lean HF and obese HF swine without appreciable changes in myocardial oxygen delivery or transmural perfusion. These findings support that high-rate ventricular pacing of lean and obese Ossabaw swine initiates underlying cardiac remodeling accompanied by elevated LV filling pressures with normal ejection fraction. This distinct pre-clinical tool provides a unique platform for further mechanistic and therapeutic studies of this highly complex syndrome.

Tgfß1-Cthrc1 Signaling Plays an Important Role in the Short-Term Reparative Response to Heart Valve Endothelial Injury.

OBJECTIVE: Aortic valve disease is a common worldwide health burden with limited treatment options. Studies have shown that the valve endothelium is critical for structure-function relationships, and disease is associated with its dysfunction, damage, or injury. Therefore, therapeutic targets to maintain a healthy endothelium or repair damaged endothelial cells could hold promise. In this current study, we utilize a surgical mouse model of heart valve endothelial cell injury to study the short-term response at molecular and cellular levels. The goal is to determine if the native heart valve exhibits a reparative response to injury and identify the mechanisms underlying this process. Approach and Results: Mild aortic valve endothelial injury and abrogated function was evoked by inserting a guidewire down the carotid artery of young (3 months) and aging (16-18 months) wild-type mice. Short-term cellular responses were examined at 6 hours, 48 hours, and 4 weeks following injury, whereas molecular profiles were determined after 48 hours by RNA-sequencing. Within 48 hours following endothelial injury, young wild-type mice restore endothelial barrier function in association with increased cell proliferation, and upregulation of transforming growth factor beta 1 (Tgfß1) and the glycoprotein, collagen triple helix repeat containing 1 (Cthrc1). Interestingly, this beneficial response to injury was not observed in aging mice with known underlying endothelial dysfunction. CONCLUSIONS: Data from this study suggests that the healthy valve has the capacity to respond to mild endothelial injury, which in short term has beneficial effects on restoring endothelial barrier function through acute activation of the Tgfß1-Cthrc1 signaling axis and cell proliferation.

Coronary remodeling and biomechanics: Are we going with the flow in 2020?

Under normal conditions, coronary blood flow (CBF) provides critical blood supply to the myocardium so that it can appropriately meet the metabolic demands of the body. Dogmatically, there exist several known regulators and modulators of CBF that include local metabolites and neurohormonal factors that can influence the function of the coronary circulation. In disease states such as diabetes and myocardial ischemia, these regulators are impaired or shifted such that CBF is reduced. Although functional considerations have been and continued to be well studied, more recent evidence builds upon established studies that collectively suggest that the relative roles of coronary structure, biomechanics, and the influence of cardiac biomechanics via extravascular compression may also play a significant role in dictating CBF. In this mini review, we discuss these regulators of CBF under normal and pathophysiological conditions and their potential influence on the control of CBF.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Mineralocorticoid receptor blockade normalizes coronary resistance in obese swine independent of functional alterations in Kv channels.

Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K+ (Kv) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO2 and potential involvement of Kv channels and whether the benefit of MR blockade involves improved coronary Kv function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine; however, baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of Kv channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary Kv function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of Kv channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.

Paired Pressure-Volume Loop Analysis and Biaxial Mechanical Testing Characterize Differences in Left Ventricular Tissue Stiffness of Volume Overload and Angiotensin-Induced Pressure Overload Hearts.

Pressure overload (PO) and volume overload (VO) of the heart result in distinctive changes to geometry, due to compensatory structural remodeling. This remodeling potentially leads to changes in tissue mechanical properties. Understanding such changes is important, as tissue modulus has an impact on cardiac performance, disease progression, and influences on cell phenotype. Pressure-volume (PV) loop analysis, a clinically relevant method for measuring left ventricular (LV) chamber stiffness, was performed in vivo on control rat hearts and rats subjected to either chronic PO through Angiotensin-II infusion (4-weeks) or VO (8-weeks). Immediately following PV loops, biaxial testing was performed on LV free wall tissue to directly measure tissue mechanical properties. The ß coefficient, an index of chamber stiffness calculated from the PV loop analysis, increased 98% in PO (n = 4) and decreased 38% in VO (n = 5) compared to control (n = 6). Material constants of LV walls obtained from ex vivo biaxial testing (n = 9-10) were not changed in Angiotensin-II induced PO and decreased by about half in VO compared to control (47% in the circumferential and 57% the longitudinal direction). PV loop analysis showed the expected increase in chamber stiffness of PO and expected decrease in chamber stiffness of VO. Biaxial testing showed a decreased modulus of the myocardium of the VO model, but no changes in the PO model, this suggests the increased chamber stiffness in PO, as shown in the PV loop analysis, may be secondary to changes in tissue mass and/or geometry but not an increase in passive tissue mechanical properties.

Reduced stiffness and augmented traction force in type 2 diabetic coronary microvascular smooth muscle.

Type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness and reduced coronary blood flow in both mice and pig models. Since reduced stiffness does not appear to be due to functional changes in the extracellular matrix, this study tested the hypothesis that decreased CRM stiffness in T2DM is due to reduced vascular smooth muscle cell (VSMC) stiffness, which impacts the traction force generated by VSMCs. Atomic force microscopy (AFM) and traction force microscopy (TFM) were conducted on primary low-passage CRM VSMCs from normal Db/db and T2DM db/db mice in addition to low-passage normal and T2DM deidentified human coronary VSMCs. Elastic modulus was reduced in T2DM mouse and human coronary VSMCs compared with normal (mouse: Db/db 6.84 ± 0.34 kPa vs. db/db 4.70 ± 0.19 kPa, P < 0.0001; human: normal 3.59 ± 0.38 kPa vs. T2DM 2.61 ± 0.35 kPa, P = 0.05). Both mouse and human T2DM coronary microvascular VSMCs were less adhesive to fibronectin compared with normal. T2DM db/db coronary VSMCs generated enhanced traction force by TFM (control 692 ± 67 Pa vs. db/db 1,507 ± 207 Pa; P < 0.01). Immunoblot analysis showed that T2DM human coronary VSMCs expressed reduced ß1-integrin and elevated ß3-integrin (control 1.00 ± 0.06 vs. T2DM 0.62 ± 0.14, P < 0.05 and control 1.00 ± 0.49 vs. T2DM 3.39 ± 1.05, P = 0.06, respectively). These data show that T2DM coronary VSMCs are less stiff and less adhesive to fibronectin but are able to generate enhanced force, corroborating previously published computational findings that decreasing cellular stiffness increases the cells' ability to generate higher traction force.NEW & NOTEWORTHY We show here that a potential causative factor for reduced diabetic coronary microvascular stiffness is the direct reduction in coronary vascular smooth muscle cell stiffness. These cells were also able to generate enhanced traction force, validating previously published computational models. Collectively, these data show that smooth muscle cell stiffness can be a contributor to overall tissue stiffness in the coronary microcirculation, and this may be a novel area of interest for therapeutic targets.

MicroRNA miR145 regulates TGFBR2 expression and matrix synthesis in vascular smooth muscle cells.

RATIONALE: MicroRNA miR145 has been implicated in vascular smooth muscle cell differentiation, but its mechanisms of action and downstream targets have not been fully defined. OBJECTIVE: Here, we sought to explore and define the mechanisms of miR145 function in smooth muscle cells. METHODS AND RESULTS: Using a combination of cell culture assays and in vivo mouse models to modulate miR145, we characterized its downstream actions on smooth muscle phenotypes. Our results show that the miR-143/145 gene cluster is induced in smooth muscle cells by coculture with endothelial cells. Endothelial cell-induced expression of miR-143/145 is augmented by Notch signaling and accordingly expression is reduced in Notch receptor-deficient cells. Screens to identify miR145-regulated genes revealed that the transforming growth factor (TGF)-ß pathway has a significantly high number of putative target genes, and we show that TGFß receptor II is a direct target of miR145. Extracellular matrix genes that are regulated by TGFß receptor II were attenuated by miR145 overexpression, and miR145 mutant mice exhibit an increase in extracellular matrix synthesis. Furthermore, activation of TGFß signaling via angiotensin II infusion revealed a pronounced fibrotic response in the absence of miR145. CONCLUSIONS: These data demonstrate a specific role for miR145 in the regulation of matrix gene expression in smooth muscle cells and suggest that miR145 acts to suppress TGFß-dependent extracellular matrix accumulation and fibrosis, while promoting TGFß-induced smooth muscle cell differentiation. Our findings offer evidence to explain how TGFß signaling exhibits distinct downstream actions via its regulation by a specific microRNA.

Coronary microvascular disease as an early culprit in the pathophysiology of diabetes and metabolic syndrome.

Metabolic syndrome (MetS) is a group of cardio-metabolic risk factors that includes obesity, insulin resistance, hypertension, and dyslipidemia; these are also a combination of independent coronary artery disease (CAD) risk factors. Alarmingly, the prevalence of MetS risk factors are increasing and a leading cause for mortality. In the vasculature, complications from MetS and type 2 diabetes (T2D) can be divided into microvascular (retinopathy and nephropathy) and macrovascular (cardiovascular diseases and erectile dysfunction). In addition to vascular and endothelial dysfunction, vascular remodeling and stiffness are also hallmarks of cardiovascular disease (CVD), and well-characterized vascular changes that are observed in the early stages of hypertension, T2D, and obesity [1-3]. In the heart, the link between obstructive atherosclerosis of coronary macrovessels and myocardial ischemia (MI) is well established. However, recent studies show that abnormalities in the coronary microcirculation are associated with functional and structural changes in coronary microvessels (classically defined as being ≤150-200µm internal diameter), which may cause or contribute to MI even in the absence of obstractive CAD. This suggests a prognostic value of an abnormal coronary microcirculation as an early sub-clinical culprit in the pathogenesis and progression of heart disease in T2D and MetS. The aim of this review is to summarize recent studies investigating the coronary microvascular remodeling in an early pre-atherosclerotic phase of MetS and T2D, and to explore potential mechanisms associated with the timing of coronary microvascular remodeling relative to that of the macrovasculature.

GPER activation ameliorates aortic remodeling induced by salt-sensitive hypertension.

The mRen2 female rat is an estrogen- and salt-sensitive model of hypertension that reflects the higher pressure and salt sensitivity associated with menopause. We previously showed that the G protein-coupled estrogen receptor (GPER) mediates estrogenic effects in this model. The current study hypothesized that GPER protects against vascular injury during salt loading. Intact mRen2 female rats were fed a normal (NS; 0.5% Na(+)) or high-salt diet (HS; 4% Na(+)) for 10 wk, which significantly increased systolic blood pressure (149 ± 5 vs. 224 ± 8 mmHg;P< 0.001). Treatment with the selective GPER agonist G-1 for 2 wk did not alter salt-sensitive hypertension (216 ± 4 mmHg;P> 0.05) or ex vivo vascular responses to angiotensin II or phenylephrine (P> 0.05). However, G-1 significantly attenuated salt-induced aortic remodeling assessed by media-to-lumen ratio (NS: 0.43; HS+veh: 0.89; HS+G-1: 0.61;P< 0.05). Aortic thickening was not accompanied by changes in collagen, elastin, or medial proliferation. However, HS induced increases in medial layer glycosaminoglycans (0.07 vs. 0.42 mm(2);P< 0.001) and lipid peroxidation (0.11 vs. 0.51 mm(2);P< 0.01), both of which were reduced by G-1 (0.20 mm(2)and 0.23 mm(2); both P< 0.05). We conclude that GPER's beneficial actions in the aorta of salt-loaded mRen2 females occur independently of changes in blood pressure and vasoreactivity. GPER-induced attenuation of aortic remodeling was associated with a reduction in oxidative stress and decreased accumulation of glycosaminoglycans. Endogenous activation of GPER may protect females from salt- and pressure-induced vascular damage.

Maternal high fat diet exposure is associated with increased hepcidin levels, decreased myelination, and neurobehavioral changes in male offspring.

Maternal obesity induces chronic inflammatory responses that impact the fetus/neonate during the perinatal period. Inflammation, iron regulation, and myelination are closely interconnected and disruptions in these processes may have deleterious effects on neurodevelopment. Hepcidin levels are increased in response to inflammation causing subsequent decreases in ferroportin and available iron needed for myelination. Our current studies were designed to test the hypotheses that: 1) maternal high fat diet (HFD) prior to and during pregnancy is sufficient to induce inflammation and alter iron regulation in the brain of the offspring, and 2) HFD exposure is associated with altered myelination and neurobehavioral deficits in the offspring. Our data revealed modest increases in inflammatory cytokines in the serum of dams fed HFD prior to pregnancy compared to dams fed a control diet (CD). Early increases in IL-5 and decreases in IL-10 were observed in serum at PN7 while IL-5 remained elevated at PN21 in the HFD-exposed pups. At PN0, most cytokine levels in whole brain homogenates were higher in the pups born to HFD-fed dams but were not different or were lower than in pups born to CD-fed dams at PN21. Conversely, the inflammation mediated transcription factor Nurr77 remained elevated at PN21. At birth, brain hepcidin, ferroportin, and l-ferritin levels were elevated in pups born to HFD-fed dams compared to pups born to CD-fed dams. Hepcidin levels remained elevated at PN7 and PN21 while ferroportin and l-ferritin levels were lower at PN7 and were not different at PN21. Decreases in myelination in the medial cortex were observed in male but not in female pups born to maternal HFD-fed dams at PN21. These structural changes correlated with changes in behavior (novel object recognition) in at 4months in males only. Our data indicate that maternal obesity (HFD) results in disruption of iron regulation in the brains of the offspring with structural and neurobehavioral deficits in males.

Enhanced Steatosis and Fibrosis in Liver of Adult Offspring Exposed to Maternal High-Fat Diet.

Early life exposures can increase the risk of developing chronic diseases including nonalcoholic fatty liver disease. Maternal high-fat diet increases susceptibility to development of steatosis in the offspring. We determined the effect of maternal high-fat diet exposure in utero and during lactation on offspring liver histopathology, particularly fibrosis. Female C57Bl/6J mice were fed a control or high-fat diet (HFD) for 8 weeks and bred with lean males. Nursing dams were continued on the same diet with offspring sacrificed during the perinatal period or maintained on either control or high-fat diet for 12 weeks. Increased hepatocyte proliferation and stellate cell activation were observed in the liver of HFD-exposed pups. Offspring exposed to perinatal high-fat diet and high-fat diet postweaning showed extensive hepatosteatosis compared to offspring on high-fat diet after perinatal control diet. Offspring exposed to perinatal high-fat diet and then placed on control diet for 12 weeks developed steatosis and pericellular fibrosis. Importantly, we found that exposure to perinatal high-fat diet unexpectedly promotes more rapid disease progression of nonalcoholic fatty liver disease, with a sustained fibrotic phenotype, only in adult offspring fed a postweaning control diet.

Effects of a myofilament calcium sensitizer on left ventricular systolic and diastolic function in rats with volume overload heart failure.

Aortocaval fistula (ACF)-induced volume overload (VO) heart failure (HF) results in progressive left ventricular (LV) dysfunction. Hemodynamic load reversal during pre-HF (4 wk post-ACF; REV) results in rapid structural but delayed functional recovery. This study investigated myocyte and myofilament function in ACF and REV and tested the hypothesis that a myofilament Ca(2+) sensitizer would improve VO-induced myofilament dysfunction in ACF and REV. Following the initial sham or ACF surgery in male Sprague-Dawley rats (200-240 g) at week 0, REV surgery and experiments were performed at weeks 4 and 8, respectively. In ACF, decreased LV function is accompanied by impaired sarcomeric shortening and force generation and decreased Ca(2+) sensitivity, whereas, in REV, impaired LV function is accompanied by decreased Ca(2+) sensitivity. Intravenous levosimendan (Levo) elicited the best inotropic and lusitropic responses and was selected for chronic oral studies. Subsets of ACF and REV rats were given vehicle (water) or Levo (1 mg/kg) in drinking water from weeks 4-8. Levo improved systolic (% fractional shortening, end-systolic elastance, and preload-recruitable stroke work) and diastolic (τ, dP/dtmin) function in ACF and REV. Levo improved Ca(2+) sensitivity without altering the amplitude and kinetics of the intracellular Ca(2+) transient. In ACF-Levo, increased cMyBP-C Ser-273 and Ser-302 and cardiac troponin I Ser-23/24 phosphorylation correlated with improved diastolic relaxation, whereas, in REV-Levo, increased cMyBP-C Ser-273 phosphorylation and increased α-to-ß-myosin heavy chain correlated with improved diastolic relaxation. We concluded that Levo improves LV function, and myofilament composition and regulatory protein phosphorylation likely play a key role in improving function.