P21-activated kinase-1 signaling is required to preserve adipose tissue homeostasis and cardiac function.

While P21-activated kinase-1 (PAK1) has been extensively studied in relation to cardiovascular health and glucose metabolism, its roles within adipose tissue and cardiometabolic diseases are less understood. In this study, we explored the effects of PAK1 deletion on energy balance, adipose tissue homeostasis, and cardiac function utilizing a whole-body PAK1 knockout (PAK1-/-) mouse model. Our findings revealed that body weight differences between PAK1-/- and WT mice emerged at 9 weeks of age, with further increases observed at 12 weeks. Furthermore, PAK1-/- mice displayed increased fat mass and decreased lean mass at 12 weeks, indicating a shift towards adiposity. In conjunction with the increased body weight, PAK1-/- mice had increased food intake and reduced energy expenditure. At a mechanistic level, PAK1 deletion boosted the expression of lipogenic markers while diminishing thermogenic markers expression in adipose tissues, contributing to reduced energy expenditure and the overall obesogenic phenotype. Moreover, our findings highlighted a significant impact on cardiac function following PAK1 deletion, including alterations in calcium kinetics and compromised systolic and lusitropy functions. In summary, our study emphasizes the significant role of PAK1 in weight regulation and cardiac function, enriching our comprehension of heart health and metabolism. These findings could potentially facilitate the identification of novel therapeutic targets in cardiometabolic diseases.

The role of P21-activated kinase (Pak1) in sinus node function.

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN). To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1-/-) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1-/- hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 µM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1-/- hearts. Reduced HCN4 expression was confirmed in Pak1-/- right atria. The reduced HCN activity in Pak1-/- could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined. Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography in preclinical rodent models, making it difficult to carry out a uniform assessment of RV function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in naïve and MCT-induced PAH rats.

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups' limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

Implications of S-glutathionylation of sarcomere proteins in cardiac disorders, therapies, and diagnosis.

The discovery that cardiac sarcomere proteins are substrates for S-glutathionylation and that this post-translational modification correlates strongly with diastolic dysfunction led to new concepts regarding how levels of oxidative stress affect the heartbeat. Major sarcomere proteins for which there is evidence of S-glutathionylation include cardiac myosin binding protein C (cMyBP-C), actin, cardiac troponin I (cTnI) and titin. Our hypothesis is that these S-glutathionylated proteins are significant factors in acquired and familial disorders of the heart; and, when released into the serum, provide novel biomarkers. We consider the molecular mechanisms for these effects in the context of recent revelations of how these proteins control cardiac dynamics in close collaboration with Ca2+ fluxes. These revelations were made using powerful approaches and technologies that were focused on thin filaments, thick filaments, and titin filaments. Here we integrate their regulatory processes in the sarcomere as modulated mainly by neuro-humoral control of phosphorylation inasmuch evidence indicates that S-glutathionylation and protein phosphorylation, promoting increased dynamics and modifying the Frank-Starling relation, may be mutually exclusive. Earlier studies demonstrated that in addition to cTnI as a well-established biomarker for cardiac disorders, serum levels of cMyBP-C are also a biomarker for cardiac disorders. We describe recent studies approaching the question of whether serum levels of S-glutathionylated-cMyBP-C could be employed as an important clinical tool in patient stratification, early diagnosis in at risk patients before HFpEF, determination of progression, effectiveness of therapeutic approaches, and as a guide in developing future therapies.

Mechanisms of troponin release into serum in cardiac injury associated with COVID-19 patients.

Serum levels of thin filament proteins, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) employing high sensitivity antibodies provide a state-of-the art determination of cardiac myocyte injury in COVID-19 patients. Although there is now sufficient evidence of the value of these determinations in patients infected with SARS-CoV-2, mechanisms of their release have not been considered in depth. We summarize the importance of these mechanisms with emphasis on their relation to prognosis, stratification, and treatment of COVID-19 patients. Apart from frank necrotic cell death, there are other mechanisms of myocyte injury leading to membrane fragility that provoke release of cTnT and cTnI. We discuss a rationale for understanding these mechanisms in COVID-19 patients with co-morbidities associated with myocyte injury such as heart failure, hypertension, arrythmias, diabetes, and inflammation. We describe how understanding these significant aspects of these mechanisms in the promotion of angiotensin signaling by SARS-CoV-2 can affect treatment options in the context of individualized therapies. Moreover, with likely omic data related to serum troponins and with the identification of elevations of serum troponins now more broadly detected employing high sensitivity antibodies, we think it is important to consider molecular mechanisms of elevations in serum troponin as an element in clinical decisions and as a critical aspect of development of new therapies.

B-arrestin-2 Signaling Is Important to Preserve Cardiac Function During Aging.

Experiments reported here tested the hypothesis that ß-arrestin-2 is an important element in the preservation of cardiac function during aging. We tested this hypothesis by aging ß-arrestin-2 knock-out (KO) mice, and wild-type equivalent (WT) to 12-16months. We developed the rationale for these experiments on the basis that angiotensin II (ang II) signaling at ang II receptor type 1 (AT1R), which is a G-protein coupled receptor (GPCR) promotes both G-protein signaling as well as ß-arrestin-2 signaling. ß-arrestin-2 participates in GPCR desensitization, internalization, but also acts as a scaffold for adaptive signal transduction that may occur independently or in parallel to G-protein signaling. We have previously reported that biased ligands acting at the AT1R promote ß-arrestin-2 signaling increasing cardiac contractility and reducing maladaptations in a mouse model of dilated cardiomyopathy. Although there is evidence that ang II induces maladaptive senescence in the cardiovascular system, a role for ß-arrestin-2 signaling has not been studied in aging. By echocardiography, we found that compared to controls aged KO mice exhibited enlarged left atria and left ventricular diameters as well as depressed contractility parameters with preserved ejection fraction. Aged KO also exhibited depressed relaxation parameters when compared to WT controls at the same age. Moreover, cardiac dysfunction in aged KO mice was correlated with alterations in the phosphorylation of myofilament proteins, such as cardiac myosin binding protein-C, and myosin regulatory light chain. Our evidence provides novel insights into a role for ß-arrestin-2 as an important signaling mechanism that preserves cardiac function during aging.

Deletion of P21-activated kinase-1 induces age-dependent increased visceral adiposity and cardiac dysfunction in female mice.

It is known that there is an age-related progression in diastolic dysfunction, especially prevalent in postmenopausal women, who develop heart failure with preserved ejection fraction (HFpEF, EF > 50%). Mechanisms and therapies are poorly understood, but there are strong correlations between obesity and HFpEF. We have tested the hypothesis that P21-activated kinase-1 (PAK1) preserves cardiac function and adipose tissue homeostasis during aging in female mice. Previous demonstrations in male mice by our lab that PAK1 activity confers cardio-protection against different stresses formed the rationale for this hypothesis. Our studies compared young (3-6 months) and middle-aged (12-15 months) female and male PAK1 knock-out mice (PAK1-/-) and wild-type (WT) equivalent. Female WT mice exhibited increased cardiac PAK1 abundance during aging. By echocardiography, compared to young WT female mice, middle-aged WT female mice showed enlargement of the left atrium as well as thickening of posterior wall and increased left ventricular mass; however, all contraction and relaxation parameters were preserved during aging. Compared to WT controls, middle-aged PAK1-/- female mice demonstrated worsening of cardiac function involving a greater enlargement of the left atrium, ventricular hypertrophy, and diastolic dysfunction. Moreover, with aging PAK1-/- female mice, unlike male PAK1-/- mice, exhibited increased adiposity with increased accumulation of visceral adipose tissue. Our data provide evidence for the significance of PAK1 signaling as an element in the preservation of cardiac function and adipose tissue homeostasis in females during aging.

Cardiac Myosin Binding Protein-C Phosphorylation Mitigates Age-Related Cardiac Dysfunction: Hope for Better Aging?

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation prevents aging-related cardiac dysfunction. We tested this hypothesis by aging genetic mouse models of hypophosphorylated cMyBP-C, wild-type equivalent, and phosphorylated-mimetic cMyBP-C for 18 to 20 months. Phosphorylated-mimetic cMyBP-C mice exhibited better survival, better preservation of systolic and diastolic functions, and unchanging wall thickness. Wild-type equivalent mice showed decreasing cMyBP-C phosphorylation along with worsening cardiac function and hypertrophy approaching those found in hypophosphorylated cMyBP-C mice. Intact papillary muscle experiments suggested that cMyBP-C phosphorylation increased cross-bridge detachment rates as the underlying mechanism. Thus, phosphorylating cMyBP-C is a novel mechanism with potential to treat aging-related cardiac dysfunction.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events.

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Prestress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.

Surgical considerations for the explantation of the Parachute left ventricular partitioning device and the implantation of the HeartMate II left ventricular assist device.

Chronic heart failure is the leading cause of death in the world. With newer therapies, the burden of this disease has decreased; however, a significant number of patients remain refractive to existing therapies. Myocardial infarction often leads to ventricular remodeling and eventually contributes to heart failure. The Parachute™ (Cardiokinetix, Menlo Park, CA) is the first device designed for percutaneous ventricular restoration therapy, which reduces left ventricular volume and minimizes the risk of open surgical procedures. For the first time, we report a case of explantation of the Parachute ventricular partitioning device and transition to a HeartMate II™ left ventricular assist device and the surgical considerations for a successful outcome.

Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in ß-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic ß-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic ß-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic ß-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and ß-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent ß-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

Successful heart transplantation using a donor heart afflicted by takotsubo cardiomyopathy.

Takotsubo cardiomyopathy, also known as apical ballooning syndrome, stress cardiomyopathy, or broken heart syndrome, is a disease characterized by transient ventricular dysfunction in the absence of obstructive coronary artery disease. Herein, we present a case in which a heart with mild takotsubo cardiomyopathy was utilized as the donor organ for an orthotopic heart transplant.

Phosphorylation of cardiac Myosin-binding protein-C is a critical mediator of diastolic function.

BACKGROUND: Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈50% of all cases of HF and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates the rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function. METHODS AND RESULTS: We compared mouse models expressing phosphorylation-deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and wild-type-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of HF. Echocardiography (ejection fraction and myocardial relaxation velocity) and pressure/volume measurements (-dP/dtmin, pressure decay time constant τ-Glantz, and passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice, whereas deficient cMyBP-C phosphorylation causes diastolic dysfunction with HFpEF in cMyBP-C(t3SA) mice. Simultaneous force and [Ca(2+)]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not because of altered [Ca(2+)]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates. CONCLUSIONS: cMyBP-C phosphorylation enhances relaxation, whereas deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or ß1-Adrenergic Stimulation.

BACKGROUND: Mammalian hearts exhibit positive inotropic responses to ß-adrenergic stimulation as a consequence of protein kinase A-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. METHODS AND RESULTS: The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca(2+)]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or ß1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca(2+)]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. CONCLUSIONS: Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with ß-adrenergic stimulation or increased pacing because of protein kinase A and CaMKIIδ phosphorylations of cMyB-C.

Cardiac myosin-binding protein-C is a critical mediator of diastolic function.

Diastolic dysfunction prominently contributes to heart failure with preserved ejection fraction (HFpEF). Owing partly to inadequate understanding, HFpEF does not have any effective treatments. Cardiac myosin-binding protein-C (cMyBP-C), a component of the thick filament of heart muscle that can modulate cross-bridge attachment/detachment cycling process by its phosphorylation status, appears to be involved in the diastolic dysfunction associated with HFpEF. In patients, cMyBP-C mutations are associated with diastolic dysfunction even in the absence of hypertrophy. cMyBP-C deletion mouse models recapitulate diastolic dysfunction despite in vitro evidence of uninhibited cross-bridge cycling. Reduced phosphorylation of cMyBP-C is also associated with diastolic dysfunction in patients. Mouse models of reduced cMyBP-C phosphorylation exhibit diastolic dysfunction while cMyBP-C phosphorylation mimetic mouse models show enhanced diastolic function. Thus, cMyBP-C phosphorylation mediates diastolic function. Experimental results of both cMyBP-C deletion and reduced cMyBP-C phosphorylation causing diastolic dysfunction suggest that cMyBP-C phosphorylation level modulates cross-bridge detachment rate in relation to ongoing attachment rate to mediate relaxation. Consequently, alteration in cMyBP-C regulation of cross-bridge detachment is a key mechanism that causes diastolic dysfunction. Regardless of the exact molecular mechanism, ample clinical and experimental data show that cMyBP-C is a critical mediator of diastolic function. Furthermore, targeting cMyBP-C phosphorylation holds potential as a future treatment for diastolic dysfunction.