Robert M. Weiss

Background: The paraventricular nucleus (PVN) of the hypothalamus is a key autonomic and cardiovascular regulatory center that contributes to neuroinflammation‐driven sympathetic excitation in heart failure. To define the cellular and transcriptional mechanisms underlying inflammatory signaling during heart failure progression, we performed single‐nucleus RNA sequencing of the PVN in rats 2 weeks after myocardial infarction (MI). Methods: PVN tissues were collected 2 weeks post MI for single‐nucleus RNA sequencing analysis. Sequencing data were processed through alignment, dimensionality reduction, clustering, and marker‐gene identification to define cell populations and gene expression profiles. Gene Set Variation Analysis and transcriptional regulatory network analyses were performed to identify altered signaling pathways and key transcription factors. Results: A total of 16 341 nuclei were classified into 5 major cell types: neurons, oligodendrocytes, astrocytes, oligodendrocyte progenitor cells, and microglia. Functional analyses identified microglia as the primary mediators of inflammatory responses in the PVN. Gene Set Variation Analysis revealed substantial pathway alterations across cell types, with microglia exhibiting marked activation of immune‐related and cytokine‐producing pathways in MI rats. Moreover, IRF5 (interferon regulatory factor 5) was identified as a master transcriptional regulator associated with inflammatory activation and was significantly upregulated in PVN microglia after MI. Increased IRF5 expression in PVN microglia was confirmed by immunofluorescence. Conclusions: Single‐nucleus RNA sequencing identified distinct cell‐specific gene signatures, regulatory networks, and signaling pathways in the PVN during heart failure, with microglial IRF5 emerging as a central regulator of immune activation and inflammatory processes. Activated IRF5 promotes microglial activation and neuroinflammation, thereby enhancing PVN neuronal activity and driving sympathetic and neurohumoral dysregulation in rats with MI. Targeting IRF5 and its downstream pathways may therefore provide new insights into the central inflammatory mechanisms contributing to cardiac dysfunction during heart failure progression.

BACKGROUND: Calcium (Ca ²⁺ ) homeostasis in cardiac fibroblasts (CFs) plays a critical role in myocardial repair and remodeling after injury. JPH2 (junctophilin-2; human JPH2 or mouse Jph2) is a structural protein known to regulate intracellular Ca ²⁺ signaling and excitation-contraction coupling in cardiomyocytes. However, the role of JPH2 in CF biology remains unexplored. METHODS: Junctophilin expression was assayed in human and mouse CFs using reverse transcription quantitative polymerase chain reaction, Western blotting, and immunofluorescence. To investigate the functional role of Jph2 in CFs, we assessed Ca ²⁺ handling with live-cell confocal imaging, conducted RNA sequencing analysis, and assayed TGFβ (transforming growth factor β) responses after adenovirus-mediated gene silencing of Jph2 and fibroblast-specific Jph2 knockout. Jph2 interactions in CFs were identified using immunoprecipitation and proximity biotin ligation assays and confirmed by coimmunoprecipitation and rescue experiments. The in vivo role of Jph2 in CFs was investigated in fibroblast-specific Jph2 knockout mice (Col1a2 ^CreERT /Jph2 ^flox/flox , Jph2 ^fKO ) at baseline and after myocardial infarction and evaluated for changes in cardiac function, fibrosis, angiogenesis, CF activation, differentiation, and proliferation. RESULTS: Jph2 was identified as the only junctophilin expressed in CFs and acutely upregulated in response to myocardial infarction. Cellular and RNA sequencing analyses revealed that isolated Jph2-deficient CFs exhibited impaired fibroblast activation, reduced extracellular matrix production, and diminished expression of VEGFA (vascular endothelial growth factor A), VEGFB, and VEGFC after TGFβ treatment. In vivo, Jph2 ^fKO mice displayed exacerbated adverse cardiac remodeling after myocardial infarction, characterized by decreased CF activation/extracellular matrix production, enhanced CF proliferation, worsened systolic dysfunction, increased left ventricular dilation, impaired scar maturation, and decreased angiogenesis. Mechanistically, biochemical assays demonstrated that Jph2 interacts directly with the coiled-coil 2 domain of Stim1 (stromal interaction molecule 1) via its joining region. Jph2 knockdown led to Stim1 protein destabilization, defective store-operated Ca ²⁺ entry, and a reduction in both canonical and noncanonical TGFβ signaling pathways. Stim1 overexpression partially rescued the sensitivity of Jph2-deficient CFs to TGFβ, as evidenced by increased expression of periostin and fibronectin-1, as well as enhanced Smad3 phosphorylation. CONCLUSIONS: Our results demonstrate that Jph2 is required in CFs to orchestrate Ca ²⁺ homeostasis, CF activation, and extracellular matrix production and promotes angiogenesis in the infarcted heart, positioning it as a central regulator of cardiac repair after injury.

Chronic overactivation of neurohormonal systems is the principal driver of adverse cardiac remodeling following myocardial infarction (MI). Recent data suggest that ablating cardiac afferent neurons in rats attenuates left ventricular (LV) remodeling following MI by blocking this overactivation. Our lab has shown that acid-sensing ion channels (ASICs) are highly expressed in cardiac afferents and may sense myocardial acidosis. We hypothesized that genetic deletion of ASICs might abrogate disadvantageous remodeling after MI by disrupting afferent signaling pathways otherwise resulting in overactivation of neurohormonal responses. To test this hypothesis, we induced MI by coronary artery ligation in wild type (WT) and ASIC3 ^-/- mice and assessed cardiac remodeling by serial echocardiography. We found that ASIC3 ^-/- mice had less LV dilation relative to MI size, increased LV mass, and increased stroke volume compared to WT mice after MI. To investigate a potential role of the autonomic nervous system, we measured renal and splanchnic sympathetic nerve activity, heart rate and systolic blood pressure variability (sBPV), and hemodynamic responses to atropine and propranolol. In addition, we assessed baroreceptor-heart rate and baroreceptor-renal sympathetic nerve activity (RSNA) reflex function. Following MI, ASIC3 ^-/- mice had lower baroreceptor-RSNA reflex sensitivity than WT mice, associated with elevated sBPV. Importantly, sBPV correlated significantly with post-MI changes in LV mass in ASIC3 ^-/- but not WT mice. Our data shows that ASIC3 plays an important role in cardiac remodeling after MI potentially via modulation of baroreflex sensitivity and sBPV. ASIC3 may be further investigated as a potential therapeutic target in heart failure.

BACKGROUND: Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca ²⁺ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca ²⁺ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. METHODS: Calpain-resistant JP2 knock-in mice (JP2 ^CR ) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2 ^CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2 ^CR , JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. RESULTS: JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2 ^CR cardiomyocytes. JP2 ^CR hearts are more resistant to pressure-overload stress, having significantly improved Ca ²⁺ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2 ^CR preserves the integrity of calpain-sensitive E-C coupling–related proteins, including ryanodine receptor 2, Ca _V 1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction–induced increases in calpain activity. Furthermore, JP2 ^CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. CONCLUSIONS: The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

Background: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood‐brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction–induced HF. Methods and Results: Male Sprague‐Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno‐associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate–dextran 10 kDa+rhodamine‐dextran 70 kDa). Compared with sham‐operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate–dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin‐1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin‐1 and tight junction–associated proteins in the PVN. Conclusions: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1^−/−) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1^−/− hearts. Although concurrent short-term KDs did not rescue cMPC1^−/− hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.