Defining a Unique Gene Expression Profile in Mature and Developing Keloids.

Keloids are benign, fibroproliferative dermal tumors that typically form owing to abnormal wound healing. The current standard of care is generally ineffective and does not prevent recurrence. To characterize keloid scars and better understand the mechanism of their formation, we performed transcriptomic profiling of keloid biopsies from a total of 25 subjects of diverse racial and ethnic origins, 15 of whom provided a paired nonlesional sample, a longitudinal sample, or both. The transcriptomic signature of nonlesional skin biopsies from subjects with keloids resembled that of control skin at baseline but shifted to closely match that of keloid skin after dermal trauma. Peripheral keloid skin and rebiopsied surrounding normal skin both showed upregulation of epithelial-mesenchymal transition markers, extracellular matrix organization, and collagen genes. These keloid signatures strongly overlapped those from healthy wound healing studies, usually with greater perturbations, reinforcing our understanding of keloids as dysregulated and exuberant wound healing. In addition, 219 genes uniquely regulated in keloids but not in normal injured or uninjured skin were also identified. This study provides insights into mature and developing keloid signatures that can act as a basis for further validation and target identification in the search for transformative keloid treatments.

Association of complement pathways with COVID-19 severity and outcomes.

OBJECTIVES: Complement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. METHODS: Longitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. RESULTS: All complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. CONCLUSION: These results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

Activin A directly impairs human cardiomyocyte contractile function indicating a potential role in heart failure development.

Activin A has been linked to cardiac dysfunction in aging and disease, with elevated circulating levels found in patients with hypertension, atherosclerosis, and heart failure. Here, we investigated whether Activin A directly impairs cardiomyocyte (CM) contractile function and kinetics utilizing cell, tissue, and animal models. Hydrodynamic gene delivery-mediated overexpression of Activin A in wild-type mice was sufficient to impair cardiac function, and resulted in increased cardiac stress markers (N-terminal pro-atrial natriuretic peptide) and cardiac atrophy. In human-induced pluripotent stem cell-derived (hiPSC) CMs, Activin A caused increased phosphorylation of SMAD2/3 and significantly upregulated SERPINE1 and FSTL3 (markers of SMAD2/3 activation and activin signaling, respectively). Activin A signaling in hiPSC-CMs resulted in impaired contractility, prolonged relaxation kinetics, and spontaneous beating in a dose-dependent manner. To identify the cardiac cellular source of Activin A, inflammatory cytokines were applied to human cardiac fibroblasts. Interleukin -1ß induced a strong upregulation of Activin A. Mechanistically, we observed that Activin A-treated hiPSC-CMs exhibited impaired diastolic calcium handling with reduced expression of calcium regulatory genes (SERCA2, RYR2, CACNB2). Importantly, when Activin A was inhibited with an anti-Activin A antibody, maladaptive calcium handling and CM contractile dysfunction were abrogated. Therefore, inflammatory cytokines may play a key role by acting on cardiac fibroblasts, causing local upregulation of Activin A that directly acts on CMs to impair contractility. These findings demonstrate that Activin A acts directly on CMs, which may contribute to the cardiac dysfunction seen in aging populations and in patients with heart failure.

Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy.

BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.

Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disorder driven by unrelenting extracellular matrix deposition. Fibroblasts are recognized as the central mediators of extracellular matrix production in IPF; however, the characteristics of the underlying fibroblast cell populations in IPF remain poorly understood. Here, we use an unbiased single-cell RNA sequencing analysis of a bleomycin-induced pulmonary fibrosis model to characterize molecular responses to fibrotic injury. Lung cells were isolated on Day 11 to capture emerging fibrosis and gene expression was analyzed by three complementary techniques, which, together, generated a 49-gene signature that defined an activated subpopulation of fibroblasts. However, none of the identified genes were specific to the activated cells or to the disease setting, implying that the activated fibroblasts are not uniquely defined, but exhibit a similar, yet amplified, gene expression pattern to control cells. Our findings have important implications for fibrosis research, including: 1) defining myofibroblasts with any single marker will fail to capture much of the underlying biology; 2) fibroblast activation is poorly correlated with expression of transforming growth factor-ß pathway genes; 3) single-cell analysis provides insight into the mechanism of action of effective therapies (nintedanib); 4) early events in lung fibrosis need not involve significant changes in fibroblast number; populations that do increase in number, such as macrophages, dendritic cells, and proliferating myeloid cells, may merit closer examination for their role in pathogenesis.

Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney Disease through Activation of LDL Receptor-Related Protein 6.

Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/ß-catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

BACKGROUND: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). DISCUSSION & CONCLUSION: This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury.

AIMS: During ischemia/reperfusion (I/R), ribosomal S6 kinase (RSK) activates Na(+)/H(+) exchanger 1 (NHE1) by phosphorylating NHE1 at serine 703 (pS703-NHE1), which promotes cardiomyocyte death and injury. Pharmacologic inhibition of NHE1 effectively protects animal hearts from I/R. However, clinical trials using NHE1 inhibitors failed to show benefit in patients with acute myocardial infarction (MI). One possible explanation is those inhibitors block both agonist-stimulated activity (increasing I/R injury) and basal NHE1 activity (necessary for cell survival). We previously showed that dominant-negative RSK (DN-RSK) selectively blocked agonist-stimulated NHE1 activity. Therefore, we hypothesized that a novel RSK inhibitor (BIX02565) would blunt agonist-stimulated NHE1 and protect hearts from I/R. METHODS AND RESULTS: Serum/angiotensin II-stimulated pS703-NHE1 was significantly decreased by BIX02565 in cultured cells. Intracellular pH recovery assay showed that BIX02565 selectively inhibited serum-stimulated NHE1 activity. Ischemia/reperfusion decreased left ventricular-developed pressure (LVDP; inhibited) to 8.7% of the basal level in non-transgenic littermate control (NLC) mouse hearts, which was significantly improved (44.6%) by BIX02565. Similar protection was observed in vehicle-treated, cardiac-specific DN-RSK-Tg mice (43%). No additional protective effect was seen in BIX02565-treated DN-RSK-Tg hearts. BIX02565 also improved LVDP in cardiac-specific wild-type (WT)-RSK-Tg mouse hearts (7.4%-40.9%, P < .01). Finally, Western Blotting results confirmed DN-RSK and BIX02565 significantly decreased I/R-induced pS703-NHE1. CONCLUSION: The RSK plays a crucial role in I/R-induced activation of NHE1 and cardiac injury. The RSK inhibition may provide an alternative target for patients with MI.

GDF11 does not rescue aging-related pathological hypertrophy.

RATIONALE: Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE: To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS: Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS: Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

Exercise training improves systolic function in hypertensive myocardium.

The general purpose of this study was to test the effect of exercise training on the left ventricular (LV) pressure-volume relationship (LV/PV) and apoptotic signaling markers in normotensive and hypertensive hearts. Four-month-old female normotensive Wistar-Kyoto rats (WKY; n = 37) and spontaneously hypertensive rats (SHR; n = 38) were assigned to a sedentary (WKY-SED, n = 21; SHR-SED, n = 19) or treadmill-trained (WKY-TRD, n = 16; SHR-TRD, n = 19) group (∼60% Vo(2 peak), 60 min/day, 5 days/wk, 12 wk). Ex vivo LV/PV were established in isovolumic Langendorff-perfused hearts, and LV levels of Akt, phosphorylated Akt (Akt(Pi)), Bad, phosphorylated Bad (Bad(Pi)) c-IAP, x-IAP, calcineurin, and caspases 3, 8, and 9 were measured. Heart-to-body weight ratio was increased in SHR vs. WKY (P < 0.05), concomitant with increased calcineurin mRNA (P < 0.05). There was a rightward shift in the LV/PV (P < 0.05) and a reduction in systolic elastance (E(s)) in SHR vs. WKY. Exercise training corrected E(s) in SHR (P < 0.05) but had no effect on the LV/PV in WKY. Caspase 3 was increased in SHR-SED relative to WKY-SED, while Bad(Pi,) c-IAP, and x-IAP were significantly lower in SHR relative to WKY (P < 0.05). Exercise training increased Bad(Pi) in both WKY and SHR but did not alter caspase 9 activity in either group. While caspase 3 activity was increased with training in WKY (P < 0.05), it was unchanged with training in SHR. We conclude that moderate levels of regular aerobic exercise attenuate systolic dysfunction early in the compensatory phase of hypertrophy, and that a differential phenotypical response to moderate-intensity exercise exists between WKY and SHR.

Cardiac ß-adrenergic responsiveness with exercise.

Left ventricular performance is enhanced with chronic exercise training. Alterations in cardiomyocyte ß-adrenergic responsiveness (BAR) may, in part, mediate this response. In this study, cardiac BAR and the expression of some key cardiac hypertrophic signaling molecules following 3 months of treadmill training were examined. Four-month old, female, Wistar Kyoto (WKY) rats were randomly assigned into either a sedentary (WKY-SED, n = 15) or an exercise-trained (WKY-TRD, n = 11) group. All rats were maintained on a 12-h light/dark cycle, and fed ad libitum. Exercise training consisted of motorized treadmill training at 25 m/min, 0% grade, 60 continuous minutes, 5 days/week for a period of 12 weeks. RT-PCR was used to establish basal cardiac calcineurin A, ANP, and AKT mRNA expression. In vitro cardiac BAR responsiveness was determined in Langendorff, isolated hearts. Following baseline, isoproterenol (ISO) was incrementally infused at concentrations ranging from 1 × 10(-10) to 1 × 10(-7) mol/L. There were no group differences for heart weight, heart to body weight ratio, calcineurin A, ANP, or AKT mRNA levels between WKY-SED and WKY-TRD. WKY-TRD showed enhanced cardiac BAR relative to WKY-SED (at ISO 1 × 10(-7) mol/L; P < 0.05). Moderate intensity treadmill exercise improved cardiac BAR responsiveness to a high concentration of isoproterenol. This adaptation was independent of training-induced alterations in cardiac hypertrophy or hypertrophic marker expression.

Increasing cardiac contractility after myocardial infarction exacerbates cardiac injury and pump dysfunction.

RATIONALE: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. OBJECTIVES: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca(2+)-handling defects could avert post-MI cardiac pump dysfunction. METHODS AND RESULTS: MI was produced in mice with inducible, cardiac-specific expression of the ß2a subunit of the L-type Ca(2+) channel. Myocyte and cardiac function were compared in control and ß2a animals before and after MI. ß2a myocytes had increased Ca(2+) current; sarcoplasmic reticulum Ca(2+) load, contraction and Ca(2+) transients (versus controls), and ß2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in ß2a versus control hearts after MI. ß2a animals also had poorer survival after MI. Myocytes isolated from ß2a hearts after MI did not develop depressed Ca(2+) handling, and Ca(2+) current, contractions, and Ca(2+) transients were still above control levels (before MI). CONCLUSIONS: Maintaining myocyte contractility after MI, by increasing Ca(2+) influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

Left ventricular remodeling with exercise in hypertension.

We investigated how exercise training superimposed on chronic hypertension impacted left ventricular remodeling. Cardiomyocyte hypertrophy, apoptosis, and proliferation in hearts from female spontaneously hypertensive rats (SHRs) were examined. Four-month-old SHR animals were placed into a sedentary group (SHR-SED; n = 18) or a treadmill running group (SHR-TRD, 20 m/min, 1 h/day, 5 days/wk, 12 wk; n = 18). Age-matched, sedentary Wistar Kyoto (WKY) rats were controls (n = 18). Heart weight was greater in SHR-TRD vs. both WKY (P < 0.01) and SHR-SED (P < 0.05). Morphometric-derived left ventricular anterior, posterior, and septal wall thickness were increased in SHR-SED relative to WKY and augmented in SHR-TRD. Cardiomyocyte surface area, length, and width were increased in SHR-SED relative to WKY and further increased in SHR-TRD. Calcineurin abundance was increased in SHR-SED vs. WKY (P < 0.001) and attenuated in SHR-TRD relative to SHR-SED (P < 0.05). Protein abundance and mRNA of Akt was not different among groups. The rate of apoptosis was increased in SHR-SED relative to WKY and mitigated in SHR-TRD. The abundance of Ki-67(+) cells across groups was not statistically different across groups. The abundance of cardiac progenitor cells (c-Kit(+) cells) was increased in SHR-TRD relative to WKY. These data suggest that exercise training superimposed on hypertension augmented cardiomyocyte hypertrophy, despite attenuating calcineurin abundance. Exercise training also mitigated apoptosis in hypertension and showed a tendency to enhance the abundance of cardiac progenitor cells, resulting in a more favorable cardiomyocyte number in the exercise-trained hypertensive heart.

CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes.

RATIONALE: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca(2+) needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca(2+) binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca(2+) handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. OBJECTIVE: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. METHODS AND RESULTS: Green fluorescent protein-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII(deltac) were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5+/-3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9+/-1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3+/-1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca(2+) increased Cn-dependent NFAT translocation (to 71.7+/-7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6+/-4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. CONCLUSION: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

Protein kinase C{alpha}, but not PKC{beta} or PKC{gamma}, regulates contractility and heart failure susceptibility: implications for ruboxistaurin as a novel therapeutic approach.

Protein kinase (PK)Calpha, PKCbeta, and PKCgamma comprise the conventional PKC isoform subfamily, which is thought to regulate cardiac disease responsiveness. Indeed, mice lacking the gene for PKCalpha show enhanced cardiac contractility and reduced susceptibility to heart failure. Recent data also suggest that inhibition of conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 enhances heart function and antagonizes failure, although the isoform responsible for these effects is unknown. Here, we investigated mice lacking PKCalpha, PKCbeta, and PKCgamma for effects on cardiac contractility and heart failure susceptibility. PKCalpha(-/-) mice, but not PKCbetagamma(-/-) mice, showed increased cardiac contractility, myocyte cellular contractility, Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load. PKCalpha(-/-) mice were less susceptible to heart failure following long-term pressure-overload stimulation or 4 weeks after myocardial infarction injury, whereas PKCbetagamma(-/-) mice showed more severe failure. Infusion of ruboxistaurin (LY333531), an orally available PKCalpha/beta/gamma inhibitor, increased cardiac contractility in wild-type and PKCbetagamma(-/-) mice, but not in PKCalpha(-/-) mice. More importantly, ruboxistaurin prevented death in wild-type mice throughout 10 weeks of pressure-overload stimulation, reduced ventricular dilation, enhanced ventricular performance, reduced fibrosis, and reduced pulmonary edema comparable to or better than metoprolol treatment. Ruboxistaurin was also administered to PKCbetagamma(-/-) mice subjected to pressure overload, resulting in less death and heart failure, implicating PKCalpha as the primary target of this drug in mitigating heart disease. As an aside, PKCalphabetagamma triple-null mice showed no defect in cardiac hypertrophy following pressure-overload stimulation. In conclusion, PKCalpha functions distinctly from PKCbeta and PKCgamma in regulating cardiac contractility and heart failure, and broad-acting PKC inhibitors such as ruboxistaurin could represent a novel therapeutic approach in treating human heart failure.

Inhibition of angiotensin II Gq signaling augments beta-adrenergic receptor mediated effects in a renal artery stenosis model of high blood pressure.

Chronic ventricular pressure overload states, such as hypertension, and elevated levels of neurohormones (norepinephrine, angiotensin II, endothelin-1) initiate cardiac hypertrophy and dysfunction and share the property of being able to bind to Gq-coupled 7-transmembrane receptors. The goal of the current study was to determine the role of endogenous cardiac myocyte Gq signaling and its role in cardiac hypertrophy and dysfunction during high blood pressure (BP). We induced renal artery stenosis for 8 weeks in control mice and mice expressing a peptide inhibitor of Gq signaling (GqI) using a 2 kidney, 1 clip renal artery stenosis model. 8 weeks following chronic high BP, control mice had cardiac hypertrophy and depressed function. Inhibition of cardiomyocyte Gq signaling did not reverse cardiac hypertrophy but attenuated increases in a profile of cardiac profibrotic genes and genes associated with remodeling. Inhibition of Gq signaling also attenuated the loss of cardiac function. We determined that Gq signaling downstream of angiotensin II receptor stimulation negatively impacted beta-adrenergic receptor (AR) responses and inhibition of Gq signaling was sufficient to restore betaAR-mediated responses. Therefore, in this study we found that Gq signaling negatively impacts cardiac function during high BP. Specifically, we found that inhibition of AT1-Gq signaling augmented betaAR mediated effects in a renal artery stenosis model of hypertension. These observations may underlie additional, beneficial effects of angiotensinogen converting enzyme (ACE) inhibitors and angiotensin receptor antagonists observed during times of hemodynamic stress.

Voluntary wheel running and pacing-induced dysfunction in hypertension.

PURPOSE: We examined how voluntary wheel running in the female, spontaneously hypertensive rat (SHR) impacts myocardial tolerance to pacing stress and determined whether direct adenylyl cyclase agonism via forskolin infusion improved myocardial performance during pacing. METHODS: Twenty-five 16-week-old female Wistar Kyoto (WKY, n = 8) and SHR (n = 17) were utilized. Animals within the SHR group were randomly assigned to a sedentary (SHR-SED, n = 8) or a voluntary wheel running (SHR-WHL, n = 9) group. The SHR-WHL had free access to a running wheel 24 h/day. Resting heart rates and blood pressures were collected immediately prior to sacrifice utilizing a tail cuff apparatus. Left ventricular (LV) function was measured in a Langendorff, isovolumic preparation during pacing stress (8.5 Hz) and during pacing stress + forskolin (5 micromol/L). RESULTS: SHR-WHL showed cardiac enlargement without alterations in heart rate, systolic blood pressure, or rate-pressure product. Pacing stress impaired inotropic and lusitropic performance to a similar extent in all groups (p < 0.05), while forskolin infusion improved LV function to a similar extent in all groups (p < 0.05). CONCLUSIONS: These data suggest that voluntary wheel running in SHR does not protect from pacing-induced myocardial dysfunction, and adenylyl cyclase agonism during pacing stress can functionally protect the heart. These data reiterate the importance of a competent myocardial beta-adrenergic signaling cascade.

Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes.

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.