The distinction between active relaxation and passive stiffness is central to understanding why the review’s cautious conclusion matters. Active relaxation is an energy-consuming process: after each heartbeat, calcium must be pumped back into the sarcoplasmic reticulum by SERCA2a, the sarco/endoplasmic reticulum calcium ATPase, and any residual calcium bound to the contractile proteins must be released so that cross-bridges detach and force declines. If calcium removal slows or myofilaments remain activated longer than they should, the ventricle begins to fill while it is still generating tension, and filling pressures rise. Passive stiffness, by contrast, is a property of the relaxed cell itself, determined largely by titin, the giant spring-like protein that spans from the Z-disc to the M-band of the sarcomere. Titin’s stiffness can be tuned by isoform switching, with the more compliant N2BA isoform giving way to the stiffer N2B isoform in some settings, and by phosphorylation of its elastic PEVK and N2B segments through kinases such as protein kinase A and protein kinase G. Hypophosphorylation of titin, oxidative modifications, and shifts in isoform ratio have all been proposed as mechanisms contributing to the elevated diastolic stiffness characteristic of HFpEF.

What makes the review’s framework particularly useful is its insistence on formal statistical testing of sex as a modifier. In experimental biology, it is common to report that females and males differ in some measured quantity, but a simple difference in a single endpoint does not establish that sex modifies the disease process. A true modifier effect requires a statistically significant interaction term, either sex by disease or sex by target, demonstrating that the effect of disease, or the response to an intervention, is itself different between the sexes. This is a much higher bar than demonstrating a baseline difference, and it is the appropriate standard when the clinical question is whether men and women should be classified or treated differently. By applying this standard systematically across the literature on calcium handling, myofilament function, titin, and mitochondrial bioenergetics, the authors converted a sprawling and often contradictory body of work into a much smaller set of defensible claims.

The result is striking in its sparseness. Among all the studies surveyed, the late sodium current emerged as the only active-relaxation-related cellular endpoint with a reported formal sex by disease interaction. The late sodium current is a small, sustained inward flow of sodium ions during the plateau phase of the cardiac action potential, and its pathological augmentation in disease states is thought to drive sodium and calcium overload through the sodium-calcium exchanger, contributing to diastolic tension and arrhythmogenic risk. The drug ranolazine, which suppresses the late current, has been tested clinically in the RALI-DHF trial for diastolic heart failure, making this target one of the few with a direct translational thread running from cellular electrophysiology to human intervention. Yet even here, the review notes that the reported sex interaction was electrophysiological rather than mechanical: the difference appeared in ionic current measurements, not in a demonstrated difference in how relaxation or stiffness responded. Without that mechanical link, the interaction remains an observation about membrane physiology rather than a guide to therapy.

The titin story illustrates a different kind of evidentiary gap. There is little doubt that titin modifications can change diastolic properties; interventions that alter titin phosphorylation or isoform expression have modified passive stiffness and filling in several HFpEF-like animal models, including models combining hypertension and metabolic stress such as the ZSF1 hybrid rat and models using L-NAME to induce nitric oxide synthase inhibition. Protein kinase G signaling, which phosphorylates titin and reduces its stiffness, has been a particularly attractive mechanistic target because it connects nitric oxide biology, cGMP signaling, and the phosphodiesterase pathways that have been explored pharmacologically in HFpEF. But the review’s systematic search found no experiment in which an independent laboratory replicated a sex-modified mechanical response to a titin-directed intervention. In other words, titin is clearly modifiable, and titin stiffness is clearly relevant to HFpEF, but the claim that women’s titin behaves differently from men’s titin in disease, in a way that would justify sex-specific treatment, has not been demonstrated under conditions that meet modern standards of rigor.

Mitochondrial bioenergetics and redox state occupy the connective position in this framework, and the review treats them accordingly. The energetic cost of relaxation is substantial: SERCA2a consumes a large fraction of the ATP generated by the cardiomyocyte, and any impairment of oxidative phosphorylation, NAD+ availability, or mitochondrial quality control can slow calcium reuptake and prolong contraction. Oxidative stress adds a second layer of vulnerability, because reactive oxygen species can modify ryanodine receptors, promote calcium leak, stiffen titin through oxidative cross-linking, and alter myofilament protein function. Pathways such as sirtuin 3, which depends on NAD+ and regulates mitochondrial protein acetylation, and calcium/calmodulin-dependent protein kinase II, which links calcium handling to oxidative stress, have each been implicated in diastolic dysfunction in experimental models. Interventions aimed at restoring NAD+ pools or reducing oxidative damage have improved diastolic phenotypes in some HFpEF-like settings. Yet here again, the review found no replicated demonstration that these bioenergetic interventions produce different mechanical benefits in females versus males, despite the plausible biological reasons to expect such differences, including known sex differences in mitochondrial function, estrogen-dependent regulation of energetics, and redox enzyme expression.

The confounding landscape surrounding this literature is unusually dense, and the review is careful to enumerate it. Findings varied with the animal model employed, the genetic strain, the age of the animals, their reproductive state, the specific disease driver used to induce the HFpEF phenotype, the stage of disease at which measurements were made, and the assay used to assess function. Each of these variables can plausibly interact with sex. Reproductive state is especially important in females, since estrogen status influences calcium handling proteins, mitochondrial enzymes, and titin phosphorylation, and ovariectomy versus intact status can change experimental outcomes. Age interacts with both sex and disease, because HFpEF is predominantly a disease of older adults and many animal models study young animals. Disease drivers matter because hypertension-driven, metabolic-driven, and anemia-driven HFpEF models produce overlapping but distinct cellular phenotypes. A sex difference observed in one combination of these variables may simply not generalize to another, which is precisely the pattern the review documents.

Human evidence, which would ideally anchor the field, is itself fragmented. Studies of ventricular tissue from patients with HFpEF have documented slowed active relaxation, elevated passive stiffness, and metabolic remodeling, including shifts in substrate utilization and mitochondrial protein expression. But these findings come from separate cohorts, often with small sample sizes, and the role of sex within each finding remains unresolved. Human myocardial tissue is difficult to obtain, typically available only from surgical procedures such as valve replacement or from transplant and autopsy material, and the underlying disease etiologies of donors vary widely. Direct comparisons of women and men using the same cellular assays on matched tissue are rare. This means that even the well-documented cellular abnormalities of HFpEF cannot currently be assigned a sex-specific prevalence or magnitude with confidence.

The review also draws a useful methodological distinction among the kinds of comparisons that would actually advance the field. One question is whether disease affects females and males differently, which requires comparing each sex against its own healthy control. A second question is whether a molecular target functions differently within diseased hearts of each sex, which requires testing the target’s contribution in both sexes under disease conditions. A third question is whether any observed difference is specific to disease rather than a baseline sex difference that exists independently of pathology. Many published studies conflate these questions, reporting sex differences without the appropriate controls to determine what kind of difference has been found. Prespecifying sex comparisons and linking them to a relevant functional endpoint, such as relaxation kinetics or passive force, would allow future studies to distinguish genuine modifier effects from incidental observations.

The clinical implications of this cautious conclusion deserve emphasis. If sex alone cannot define calcium-dominant or titin-dominant HFpEF endotypes, then treatment selection based on a patient’s sex would currently lack a mechanistic foundation. This does not mean sex is irrelevant to HFpEF; the epidemiological preponderance of women, differences in ventricular remodeling patterns, and known hormonal influences on cardiovascular physiology all remain real and clinically important. It means instead that the cellular mechanisms underlying any sex-related clinical differences have not been pinned down with the specificity needed to guide endotyping. The practical path forward suggested by the review is to treat sex as a hypothesis-generating variable to be tested in defined experimental and clinical settings, rather than as a classification tool. Until replicated, sex-stratified, mechanically anchored evidence accumulates, the heterogeneous reality of HFpEF, in which multiple cellular defects coexist and vary with context, remains the best available description of the disease for both women and men.

Subject of Research: Sex differences in cardiomyocyte relaxation mechanisms in heart failure with preserved ejection fraction

Article Title: Evaluating sex as a modifier of cardiomyocyte relaxation in heart failure with preserved ejection fraction: calcium handling, titin, and bioenergetics

Article References: Wang, B., & Dai, X.-C. (2026). Evaluating sex as a modifier of cardiomyocyte relaxation in heart failure with preserved ejection fraction: calcium handling, titin, and bioenergetics. Biology of Sex Differences. https://doi.org/10.1186/s13293-026-00993-8

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00993-8

Keywords: HFpEF, cardiomyocyte relaxation, calcium handling, titin, passive stiffness, mitochondrial bioenergetics, sex differences, late sodium current, NAD+, diastolic dysfunction, heart failure, Biology of Sex Differences