Guidelines on the use of sex and gender in cardiovascular research.

In cardiovascular research, sex and gender have not typically been considered in research design and reporting until recently. This has resulted in clinical research findings from which not only all women, but also gender-diverse individuals have been excluded. The resulting dearth of data has led to a lack of sex- and gender-specific clinical guidelines and raises serious questions about evidence-based care. Basic research has also excluded considerations of sex. Including sex and/or gender as research variables not only has the potential to improve the health of society overall now, but it also provides a foundation of knowledge on which to build future advances. The goal of this guidelines article is to provide advice on best practices to include sex and gender considerations in study design, as well as data collection, analysis, and interpretation to optimally establish rigor and reproducibility needed to inform clinical decision-making and improve outcomes. In cardiovascular physiology, incorporating sex and gender is a necessary component when optimally designing and executing research plans. The guidelines serve as the first guidance on how to include sex and gender in cardiovascular research. We provide here a beginning path toward achieving this goal and improve the ability of the research community to interpret results through a sex and gender lens to enable comparison across studies and laboratories, resulting in better health for all.

The effect of gradual ovarian failure on dynamic muscle function and the role of high-intensity interval training on mitigating impairments.

Skeletal muscle contractile function is impaired in menopause and exercise may mitigate this decline. We used the 4-vinylcyclohexene diepoxide (VCD) model of menopause to investigate the effects of gradual ovarian failure on skeletal muscle contractile function and whether high-intensity interval training (HIIT) can mitigate impairments. Sexually mature female CD-1 mice were assigned to one of three groups: control sedentary (n = 5), VCD-sedentary (n = 5), or VCD-training (n = 5). Following ovarian failure (a 4-mo process), the VCD-training group underwent 8 wk of uphill HIIT. Mice were euthanized 8 wk after ovarian failure, representing late menopause. Single fibers from the soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected, chemically permeabilized, and mechanically tested. Single muscle fibers were maximally activated (pCa 4.5), then isotonic load clamps were performed to evaluate force-velocity-power relationships. Absolute force and peak power were 31.0% and 32.2% lower in VCD-sedentary fibers compared with control fibers, respectively, in both SOL and EDL muscles. Despite reductions in absolute force, there were no concomitant increases in contractile velocity to preserve power production. HIIT attenuated force loss in the VCD-training group such that peak force was not different from the control group across muscles and was partially effective at mitigating power loss (21.7% higher peak power in VCD-training compared with VCD-sedentary) but only in fast-type SOL fibers. These findings indicate that ovarian failure impairs dynamic contractile function-likely through a combination of lower force-generating capacity and slower shortening velocity-and that HIIT may be insufficient to completely counteract the deleterious effects of menopause at the cellular level.NEW & NOTEWORTHY We used the VCD model of menopause to investigate the effects of gradual ovarian failure on skeletal muscle contractile function and whether high-intensity interval training (HIIT) can mitigate impairments. Our findings indicate that ovarian failure impairs dynamic contractile function-likely through a combination of lower force-generating capacity and slower shortening velocity-and that HIIT may be insufficient to completely counteract the deleterious effects of menopause at the cellular level.

Sex differences in the cardiac stress response following SARS-CoV-2 infection of ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection damages the heart, increasing the risk of adverse cardiovascular events. Female sex protects against complications of infection; females are less likely to experience severe illness or death, although their risk for postacute sequelae of COVID-19 ("long COVID") is higher than in males. Despite the important role of the heart in COVID-19 outcomes, molecular elements in the heart impacted by SARS-CoV-2 are poorly understood. Similarly, the role sex has on the myocardial effects of SARS-CoV-2 infection has not been investigated at a molecular level. We intranasally inoculated female and male ferrets with SARS-CoV-2 and assessed myocardial stress signals, inflammation, and the innate immune response for 14 days. Myocardial phosphorylated GSK3α/ß decreased at day 2 postinfection (pi) in male ferrets, whereas females showed no changes. Myocardial levels of p62/SQSTM1 decreased in male ferrets at days 2, 7, and 14 pi while lower baseline levels in females increased on day 2. Phosphorylated ERK1/2 increased in cardiomyocyte nuclei in females on days 2 and 14 pi, whereas male ferrets had no changes. Only hearts from females increased fibrosis on day 14 pi. Immune and inflammation markers increased in hearts, with some sex differences. These results are the first to identify myocardial stress responses following SARS-CoV-2 infection and reveal sex differences that may contribute to differential outcomes. Future research is required to define the pathways involving these stress signals to fully understand the myocardial effects of COVID-19 and identify targets that mitigate cardiac injury following SARS-CoV-2 infection.NEW & NOTEWORTHY Cardiovascular disease is a leading risk factor for severe COVID-19, and cardiovascular pathologies are among the most common adverse outcomes following SARS-CoV-2 infection. Females and males have different outcomes and adverse cardiovascular events following SARS-CoV-2 infection. This study shows sex differences in stress proteins p62/SQSTM1, ERK1/2, and GSK3α/ß, along with innate immunity and inflammation in hearts of ferrets infected with SARS-CoV-2, identifying mechanisms of COVID-19 cardiac injury and cardiac complications of long COVID.

Predominant myosin superrelaxed state in canine myocardium with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM.NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

Recalibrating healthcare to create a more equitable post-pandemic work environment.

The COVID-19 pandemic has accelerated the need for flexible arrangements, including asynchronous work and working from home. These arrangements may be necessary to comply with public health directives and are manageable when few other options exist. It can be difficult to lead in an environment when team members have divergent core working hours and are not available for collaboration. This can be compounded by the perception of inequitable treatment of employee needs or preferences by management, which can further strain team dynamics. As the pandemic eases, it may be difficult for all employees to revert to a fully on-site arrangement; some may be unable and others unwilling. Leaders will need to consider ethical issues in reaching organizational goals in this new reality. Equity, diversity, and inclusion principles will be critical when balancing the needs of the individual and the team. Supportive arrangements and a culture of inclusion will be key to retaining top talent.

Remote ischemic conditioning for acute respiratory distress syndrome in COVID-19.

Acute respiratory distress syndrome and subsequent respiratory failure remains the leading cause of death (>80%) in patients severely impacted by COVID-19. The lack of clinically effective therapies for COVID-19 calls for the consideration of novel adjunct therapeutic approaches. Though novel antiviral treatments and vaccination hold promise in control and prevention of early disease, it is noteworthy that in severe cases of COVID-19, addressing "run-away" inflammatory cascades are likely more relevant for improvement of clinical outcomes. Viral loads may decrease in severe, end-stage coronavirus cases, but a systemically damaging cytokine storm persists and mediates multiple organ injury. Remote ischemic conditioning (RIC) of the limbs has shown potential in recent years to protect the lungs and other organs against pathological conditions similar to that observed in COVID-19. We review the efficacy of RIC in protecting the lungs against acute injury and current points of consideration. The beneficial effects of RIC on lung injury along with other related cardiovascular complications are discussed, as are the limitations presented by sex and aging. This adjunct therapy is highly feasible, noninvasive, and proven to be safe in clinical conditions. If proven effective in clinical trials for acute respiratory distress syndrome and COVID-19, application in the clinical setting could be immediately implemented to improve outcomes.

Long-term testosterone deficiency modifies myofilament and calcium-handling proteins and promotes diastolic dysfunction in the aging mouse heart.

The impact of long-term gonadectomy (GDX) on cardiac contractile function was explored in the setting of aging. Male mice were subjected to bilateral GDX or sham operation (4 wk) and investigated at 16-18 mo of age. Ventricular myocytes were field stimulated (2 Hz, 37°C). Peak Ca2+ transients (fura 2) and contractions were similar in GDX and sham-operated mice, although Ca2+ transients (50% decay time: 45.2 ± 2.3 vs. 55.6 ± 3.1 ms, P < 0.05) and contractions (time constant of relaxation: 39.1 ± 3.2 vs. 69.5 ± 9.3 ms, P < 0.05) were prolonged in GDX mice. Action potential duration was increased in myocytes from GDX mice, but this did not account for prolonged responses, as Ca2+ transient decay was slow even when cells from GDX mice were voltage clamped with simulated "sham" action potentials. Western blots of proteins involved in Ca2+ sequestration and efflux showed that Na+/Ca2+ exchanger and sarco(endo)plasmic reticulum Ca2+-ATPase type 2 protein levels were unaffected, whereas phospholamban was dramatically higher in ventricles from aging GDX mice (0.24 ± 0.02 vs. 0.86 ± 0.13, P < 0.05). Myofilament Ca2+ sensitivity at physiological Ca2+ was similar, but phosphorylation of essential myosin light chain 1 was reduced by ≈50% in ventricles from aging GDX mice. M-mode echocardiography showed no change in systolic function (e.g., ejection fraction). Critically, pulse-wave Doppler echocardiography showed that GDX slowed isovolumic relaxation time (12.9 ± 0.9 vs. 16.9 ± 1.0 ms, P < 0.05), indicative of diastolic dysfunction. Thus, dysregulation of intracellular Ca2+ and myofilament dysfunction contribute to deficits in contraction in hearts from testosterone-deficient aging mice. This suggests that low testosterone helps promote diastolic dysfunction in the aging heart. NEW & NOTEWORTHY The influence of long-term gonadectomy on contractile function was examined in aging male hearts. Gonadectomy slowed the decay of Ca2+ transients and contractions in ventricular myocytes and slowed isovolumic relaxation time, demonstrating diastolic dysfunction. Underlying mechanisms included Ca2+ dysregulation, elevated phospholamban protein levels, and hypophosphorylation of a myofilament protein, essential myosin light chain. Testosterone deficiency led to intracellular Ca2+ dysregulation and myofilament dysfunction, which may facilitate diastolic dysfunction in the setting of aging.

Acute exposure to progesterone attenuates cardiac contraction by modifying myofilament calcium sensitivity in the female mouse heart.

Acute application of progesterone attenuates cardiac contraction, although the underlying mechanisms are unclear. We investigated whether progesterone modified contraction in isolated ventricular myocytes and identified the Ca2+ handling mechanisms involved in female C57BL/6 mice (6-9 mo; sodium pentobarbital anesthesia). Cells were field-stimulated (4 Hz; 37°C) and exposed to progesterone (0.001-10.0 µM) or vehicle (35 min). Ca2+ transients (fura-2) and cell shortening were recorded simultaneously. Maximal concentrations of progesterone inhibited peak contraction by 71.4% (IC50 = 160 ± 50 nM; n = 12) and slowed relaxation by 75.4%. By contrast, progesterone had no effect on amplitudes or time courses of underlying Ca2+ transients. Progesterone (1 µM) also abbreviated action potential duration. When the duration of depolarization was controlled by voltage-clamp, progesterone attenuated contraction and slowed relaxation but did not affect Ca2+ currents, Ca2+ transients, sarcoplasmic reticulum (SR) content, or fractional release of SR Ca2+ Actomyosin MgATPase activity was assayed in myofilaments from hearts perfused with progesterone (1 µM) or vehicle (35 min). While maximal responses to Ca2+ were not affected by progesterone, myofilament Ca2+ sensitivity was reduced (EC50 = 0.94 ± 0.01 µM for control, n = 7 vs. 1.13 ± 0.05 µM for progesterone, n = 6; P < 0.05) and progesterone increased phosphorylation of myosin binding protein C. The effects on contraction were inhibited by lonaprisan (progesterone receptor antagonist) and levosimendan (Ca2+ sensitizer). Unlike results in females, progesterone had no effect on contraction or myofilament Ca2+ sensitivity in age-matched male mice. These data indicate that progesterone reduces myofilament Ca2+ sensitivity in female hearts, which may exacerbate manifestations of cardiovascular disease late in pregnancy when progesterone levels are high. NEW & NOTEWORTHY: We investigated myocardial effects of acute application of progesterone. In females, but not males, progesterone attenuates and slows cardiomyocyte contraction with no effect on calcium transients. Progesterone also reduces myofilament calcium sensitivity in female hearts. This may adversely affect heart function, especially when serum progesterone levels are high in pregnancy.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/acute-progesterone-modifies-cardiac-contraction/.

The neglected messengers: Control of cardiac myofilaments by protein phosphatases.

Cardiac myofilaments act as the central contractile apparatus of heart muscle cells. Covalent modification of constituent proteins through phosphorylation is a rapid and powerful mechanism to control myofilament function, and is increasingly seen as a mechanism of disease. While the relationship between protein kinases and cardiac myofilaments has been widely examined, the impact of protein dephosphorylation by protein phosphatases is poorly understood. This review outlines the mechanisms by which the mostly widely expressed protein phosphatases in cardiac myocytes regulate myofilament function, and the emerging role of myofilament-associated protein phosphatases in heart failure. The importance of regulatory subunits and subcellular compartmentalization in determining the functional impact of protein phosphatases on myofilament and myocardial function is also discussed, as are discrepancies about the roles of protein phosphatases in regulating myofilament function. The potential for targeting these molecular messengers in the treatment of heart failure is discussed as a key future direction.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.

Day-night dependence of gene expression and inflammatory responses in the remodeling murine heart post-myocardial infarction.

Diurnal or circadian rhythms are fundamentally important for healthy cardiovascular physiology and play a role in timing of onset and tolerance to myocardial infarction (MI) in patients. Whether time of day of MI triggers different molecular and cellular responses that can influence myocardial remodeling is not known. This study was designed to test whether time of day of MI triggers different gene expression, humoral, and innate inflammatory responses that contribute to cardiac repair after MI. Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 2-h time window either shortly after lights on or lights off, and the early remodeling responses at 8 h postinfarction were examined. We found that sleep-MI preferentially triggers early expression of genes associated with inflammatory responses, whereas wake-MI triggers more genes associated with metabolic pathways and transcription/translation, by microarray analyses. Homozygous clock mutant mice exhibit altered diurnal gene expression profiles, consistent with their cycling before onset of MI. In the first 8 h, crucial for innate immune responses to MI, there are also significant differences in sleep-MI and wake-MI serum cytokine responses and in neutrophil infiltration to infarcted myocardium. By 1-wk post-MI, there are differences in survivorship between the sleep and wake MI mice that could be explained by the different molecular and cellular responses. Our whole body physiology, tissues, and cells exhibit endogenous daily rhythms, and understanding their role in triggering effective responses after MI could lead to new strategies to benefit patients with cardiovascular disease.

Short-term disruption of diurnal rhythms after murine myocardial infarction adversely affects long-term myocardial structure and function.

RATIONALE: Patients in intensive care units are disconnected from their natural environment. Synchrony between environmental diurnal rhythms and intracellular circadian rhythms is essential for normal organ biology; disruption causes pathology. Whether disturbing rhythms after myocardial infarction (MI) exacerbates long-term myocardial dysfunction is not known. OBJECTIVE: Short-term diurnal rhythm disruption immediately after MI impairs remodeling and adversely affects long-term cardiac structure and function in a murine model. METHODS AND RESULTS: Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 3-hour time window, randomized to either a normal diurnal or disrupted environment for 5 days, and then maintained under normal diurnal conditions. Initial infarct size was identical. Short-term diurnal disruption adversely affected body metabolism and altered early innate immune responses. In the first 5 days, crucial for scar formation, there were significant differences in cardiac myeloperoxidase, cytokines, neutrophil, and macrophage infiltration. Homozygous clock mutant mice exhibited altered infiltration after MI, consistent with circadian mechanisms underlying innate immune responses crucial for scar formation. In the proliferative phase, 1 week after MI, this led to significantly less blood vessel formation in the infarct region of disrupted mice; by day 14, echocardiography showed increased left ventricular dilation and infarct expansion. These differences continued to evolve with worse cardiac structure and function by 8 weeks after MI. CONCLUSIONS: Diurnal rhythm disruption immediately after MI impaired healing and exacerbated maladaptive cardiac remodeling. These preclinical findings suggest that disrupted diurnal rhythms such as found in modern intensive care unit environments may adversely affect long-term patient outcome.

Cardiac contraction, calcium transients, and myofilament calcium sensitivity fluctuate with the estrous cycle in young adult female mice.

This study established conditions to induce regular estrous cycles in female C57BL/6J mice and investigated the impact of the estrous cycle on contractions, Ca2+ transients, and underlying cardiac excitation-contraction (EC)-coupling mechanisms. Daily vaginal smears from group-housed virgin female mice were stained to distinguish estrous stage (proestrus, estrus, metestrus, diestrus). Ventricular myocytes were isolated from anesthetized mice. Contractions and Ca2+ transients were measured simultaneously (4 Hz, 37 °C). Interestingly, mice did not exhibit regular cycles unless they were exposed to male pheromones in bedding added to their cages. Field-stimulated myocytes from mice in estrus had larger contractions (∼2-fold increase), larger Ca2+ transients (∼1.11-fold increase), and longer action potentials (>2-fold increase) compared with other stages. Larger contractions and Ca2+ transients were not observed in estrus myocytes voltage-clamped with shorter action potentials. Voltage-clamp experiments also demonstrated that estrous stage had no effect on Ca2+ current, EC-coupling gain, diastolic Ca2+, sarcoplasmic reticulum (SR) Ca2+ content, or fractional release. Although contractions were largest in estrus, myofilament Ca2+ sensitivity was lowest (EC50 values ∼1.15-fold higher) in conjunction with increased phosphorylation of myosin binding protein C in estrus. Contractions were enhanced in ventricular myocytes from mice in estrus because action potential prolongation increased SR Ca2+ release. These findings demonstrate that cyclical changes in reproductive hormones associated with the estrous cycle can influence myocardial electrical and contractile function and modify Ca2+ homeostasis. However, such changes are unlikely to occur in female mice housed in groups under conventional conditions, since these mice do not exhibit regular estrous cycles.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Cold acclimation increases cardiac myofilament function and ventricular pressure generation in trout.

Reducing temperature below the optimum of most vertebrate hearts impairs contractility and reduces organ function. However, a number of fish species, including the rainbow trout, can seasonally acclimate to low temperature. Such ability requires modification of physiological systems to compensate for the thermodynamic effects of temperature on biological processes. The current study tested the hypothesis that rainbow trout compensate for the direct effect of cold temperature by increasing cardiac contractility during cold acclimation. We examined cardiac contractility, following thermal acclimation (4, 11 and 17°C), by measuring the Ca(2+) sensitivity of force generation by chemically skinned cardiac trabeculae as well as ventricular pressure generation using a modified Langendorff preparation. We demonstrate, for the first time, that the Ca(2+) sensitivity of force generation was significantly higher in cardiac trabeculae from 4°C-acclimated trout compared with those acclimated to 11 or 17°C, and that this functional change occurred in parallel with a decrease in the level of cardiac troponin T phosphorylation. In addition, we show that the magnitude and rate of ventricular pressure generation was greater in hearts from trout acclimated to 4°C compared with those from animals acclimated to 11 or 17°C. Taken together, these results suggest that enhanced myofilament function, caused by modification of existing contractile proteins, is at least partially responsible for the observed increase in pressure generation after acclimation to 4°C. In addition, by examining the phenotypic plasticity of a comparative model we have identified a strategy, used in vivo, by which the force-generating capacity of cardiac muscle can be increased.

Effects of chemically induced ovarian failure on single muscle fiber contractility in a mouse model of menopause.

OBJECTIVE: Menopause is associated with impaired skeletal muscle contractile function. The temporal and mechanistic bases of this dysfunction are unknown. Using a mouse model of menopause, we identified how gradual ovarian failure affects single muscle fiber contractility. STUDY DESIGN: Ovarian failure was chemically induced over 120 days, representing the perimenopausal transition. Mice were sacrificed and soleus and extensor digitorum longus muscles were dissected and chemically permeabilized for single fiber mechanical testing. MAIN OUTCOME MEASURES: Muscle fiber contractility was assessed via force, rate of force redevelopment, instantaneous stiffness, and calcium sensitivity. RESULTS: Peak force and cross-sectional area of the soleus were, respectively, ~33 % and ~24 % greater following ovarian failure compared with controls (p < 0.05) with no differences in force produced by the extensor digitorum longus across groups (p > 0.05). Upon normalizing force to cross-sectional area there were no differences across groups (p > 0.05). Following ovarian failure, rate of force redevelopment of single fibers from the soleus was ~33 % faster compared with controls. There was no shift in the midpoint of the force­calcium curve between groups or muscles (p > 0.05). However, following ovarian failure, Type I fibers from the soleus had a higher calcium sensitivity between pCa values of 4.5 and 6.2 compared with controls (p < 0.05), with no differences for Type II fibers or the extensor digitorum longus (p > 0.05). CONCLUSIONS: In our model of menopause, alterations to muscle contractility were less evident than in ovariectomized models. This divergence across models highlights the importance of better approximating the natural trajectory of menopause during and after the transitional phase of ovarian failure on neuromuscular function.

Perimenopause Decreases SERCA2a Activity in the Hearts of a Mouse Model of Ovarian Failure.

Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium-calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.

Low molecular weight fibroblast growth factor-2 signals via protein kinase C and myofibrillar proteins to protect against postischemic cardiac dysfunction.

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV(1-2)) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R (P < 0.05), which was significantly abrogated in the absence of PKCα (P < 0.05) or presence of PKCε inhibition (P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction.

Innovative Animal Models Of Cardiac Remodeling: Development And Evaluation.

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