Heart failure with preserved ejection fraction (HFpEF) is a widespread syndrome with limited therapeutic options and poorly understood immune pathophysiology. Using a 2-hit preclinical model of cardiometabolic HFpEF that induces obesity and hypertension, we found that cardiac T cell infiltration and lymphoid expansion occurred concomitantly with cardiac pathology and that diastolic dysfunction, cardiomyocyte hypertrophy, and cardiac phospholamban phosphorylation were T cell dependent. Heart-infiltrating T cells were not restricted to cardiac antigens and were uniquely characterized by impaired activation of the inositol-requiring enzyme 1α/X-box–binding protein 1 (IRE1α/XBP1) arm of the unfolded protein response. Notably, selective ablation of XBP1 in T cells enhanced their persistence in the heart and lymphoid organs of mice with preclinical HFpEF. Furthermore, T cell IRE1α/XBP1 activation was restored after withdrawal of the 2 comorbidities inducing HFpEF, resulting in partial improvement of cardiac pathology. Our results demonstrated that diastolic dysfunction and cardiomyocyte hypertrophy in preclinical HFpEF were T cell dependent and that reversible dysregulation of the T cell IRE1α/XBP1 axis was a T cell signature of HFpEF.

Background: Despite the well-established association between T-cell–mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)–modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. Methods: We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77 ^GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation–deficient MhcII ^−/− mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. Results: We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII ^−/− mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction–induced cardiac dysfunction despite the presence of left ventricle–infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species–dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. Conclusions: Our study demonstrates an important role of reactive oxygen species–induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Background: Cyclic GMP ( cGMP ) signaling attenuates cardiac remodeling, but it is unclear which cGMP effectors mediate these effects and thus might serve as novel therapeutic targets. Therefore, we tested whether the cGMP downstream effector, cGMP ‐dependent protein kinase G Iα ( PKG Iα), attenuates pressure overload–induced remodeling in vivo. Methods and Results: The effect of transaortic constriction ( TAC )–induced left ventricular ( LV ) pressure overload was examined in mice with selective mutations in the PKG Iα leucine zipper interaction domain. Compared with wild‐type littermate controls, in response to TAC , these L eucine Z ipper M utant ( LZM ) mice developed significant LV systolic and diastolic dysfunction by 48 hours (n=6 WT sham, 6 WT TAC , 5 LZM sham, 9 LZM TAC ). In response to 7‐day TAC , the LZM mice developed increased pathologic hypertrophy compared with controls (n=5 WT sham, 4 LZM sham, 8 WT TAC , 11 LZM TAC ). In WT mice, but not in LZM mice, phosphodiesterase 5 ( PDE 5) inhibition with sildenafil ( Sil ) significantly inhibited TAC ‐induced cardiac hypertrophy and LV systolic dysfunction in WT mice, but this was abolished in the LZM mice (n=3 WT sham, 4 LZM sham, 3 WT TAC vehicle, 6 LZM TAC vehicle, 4 WT TAC Sil , 6 LZM TAC Sil ). And in response to prolonged, 21‐day TAC (n=8 WT sham, 7 LZM sham, 21 WT TAC , 15 LZM TAC ), the LZM mice developed markedly accelerated mortality and congestive heart failure. TAC induced activation of JNK , which inhibits cardiac remodeling in vivo, in WT , but not in LZM , hearts, identifying a novel signaling pathway activated by PKG Iα in the heart in response to LV pressure overload. Conclusions: These findings reveal direct roles for PKG Iα in attenuating pressure overload–induced remodeling in vivo and as a required effector for the cardioprotective effects of sildenafil.

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.

Background—: Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. Methods and Results—: We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ)–binding domain to identify myocardial-specific PKGI antiremodeling substrates. Our screen identified cardiac myosin–binding protein-C (cMyBP-C), a cardiac myocyte–specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα–cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Conclusions—: Taken together, these data support that PKGIα and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial-specific PKGIα LZ-dependent antiremodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.