A total of 80 male 8-week-old Wistar rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 208 ± 14 g, were used for this study. Rats were housed at standard SPF laboratory and free access to standard rodent chow and water. Randomly, 30 rats were divided into sham (sham group, n = 15) and sham with inhalation of zinc particles (sham-Zn group, n = 15) and 50 rats were performed with left anterior descending artery (LAD) ligation surgery to induce MI. Six animals died during the surgery. Three days after surgery, half of the surviving MI rats (n = 22) were exposed in the environment filled with ultrafine zinc particle (diameter of 50 nm and density of 500 μg/m³) (MI-Zn group, n = 22) using the same method as a previous study (Bing et al., 2020) while the rest were considered as the MI group (n = 22). MI-Zn and Sham-Zn rats inhaled ultrafine zinc particles for 4 h per day and 4 days per week for 4 weeks, as shown in Figure 1A. All experiments were performed in accordance with Chinese National and Peking University ethical guidelines regarding the use of animals in research, consistent with the NIH guidelines (Guide for the care and use of laboratory animals) on protection of animals used for scientific purposes. The experimental protocol was approved by the Animal Care and Use Committee of Peking University, China.

Left anterior descending artery ligation was performed in 50 Wistar rats. Briefly, in sterile environment, surgical anesthesia was maintained with ∼2% isoflurane and animals were intubated and ventilated with room air and oxygen using a Harvard ventilator (Inspira) (Niu et al., 2020). After the chest was shaved and sterilized, a left thoracotomy was performed between the third and fourth intercostal spaces. A 7-0 suture line was placed at 1 mm below the left auricle appendage to ligate the LAD artery, which led to pale LV anterior wall and apex region. Alternatively, the suture was placed but removed in sham-operated animals. After the chest was closed, animals were intramuscularly administered a dose of penicillin (400,000 U) and 1 ml dezocine (50 μg/ml) and allowed to recover from the surgery. Animals were given an intramuscular injection of penicillin (400,000 U) and 1 ml dezocine (50 μg/ml) for three consecutive days. All animals were cared at 26°C indoors, and under 12:12 h light/dark artificial cycle conditions for a total of 4 weeks after surgical recovery.

Echocardiographic measurements of rat hearts, as shown in Figure 1B, were carried out under anesthesia for 4 weeks postoperatively. M-mode measurements of LV, left atrium, and aorta and B-mode measurements of strain and strain rate were recorded in rats, similar to a previous study (Niu et al., 2020). The images were obtained at 21 MHz using a MS-250 transducer operated by a Vevo 2100 Color Doppler Ultrasound Scanner (FUJIFILM VisualSonics Inc.). Based on M-mode tracings, morphometric parameters, e.g., LVID;d, LVID;s, LVFW;s, LVFW;d, IVS;s, and IVS;d, were measured according to the American Society of Echocardiography leading edge rule (Sahn et al., 1978). These parameters were averaged based on five measurements. Moreover, FS (%) and EF (%) were calculated from the measured parameters as: (L⁢V⁢I⁢D;d-L⁢V⁢I⁢D;s)L⁢V⁢I⁢D;d×100% and (L⁢V⁢I⁢D;d3-L⁢V⁢I⁢D;s3)L⁢V⁢I⁢D;d3×100%, respectively, on a Vevo LAB image analysis workstation.

Myocardial deformation and movement measurements were carried out by using the Vevo LAB image analysis workstation with advanced STE, which tracks natural acoustic markers (called speckles) across the cardiac cycle and estimates velocity vectors. Strain measurement of myocardial deformation were obtained from B-Mode cine loops acquired from the parasternal long-axis and short-axis views (Niu et al., 2020). Frame rate is 133 Hz, gain is 20∼25 dB, depth is ∼20 mm, width is ∼23 mm, and three cardiac cycles were recorded. Longitudinal and circumferential stains (S = Δ⁢LL0, where L₀ and ΔL refer to the baseline length at the R-Wave and the absolute change in length, respectively) and strain rate (SR = ⁢SΔ⁢t = ⁢(Δ⁢L/L0)Δ⁢t = (Δ⁢L/Δ⁢t)L0= Δ⁢VL0, where ΔV is the velocity gradient in the segment) were determined by the software across a selected period of cardiac cycles.

The left carotid artery (LCA) was dissected in a sterile environment under anesthesia after the echocardiographic measurements. A perivascular flow probe (Transonic Systems Inc.; relative error of ±2% at full scale) was used to measure volumetric flow rate of LCA. Moreover, a 1.4F micromanometer-tipped catheter (Millar Instruments) was inserted through the right carotid artery into the LV to record pressure waves over 30 cardiac cycles, which was repeated three times. The zero-pressure baseline of the catheter was calibrated in the 37°C saline solution. The catheter and perivascular flow probe were monitored with a BIOPAC MP150. Heart rate, LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and rate of maximum positive and negative left ventricular pressure development (dpdtmax and dpdtmin) were determined from the measured pressure waves.

After hemodynamic measurements, blood samples were taken from the tail vein and centrifuged at 3000 RPM for 15 min. The serum was extracted and stored at −20°C. The Blood Zinc Concentration Detection Kit (Solarbio BC2815, Beijing Solarbio Science & Technology Co., Ltd.) was used to detect zinc concentration in the serum, which was measured by the microplate reader (Multiskan^TM FC, Thermo Fisher Scientific, United States) with a wavelength of 620 nm.

All animals were terminated for the histological analysis at postoperative 4 weeks by intraperitoneal injection of 1% pentobarbital sodium at dose of 150 mg/kg (Niu et al., 2020). After hearts were harvested, plugs of myocardial tissues were removed from different positions of the LV. These plugs were fixed in 4% paraformaldehyde (PFA)/PBS solution overnight at room temperature and then processed for paraffin sectioning. Masson’s trichrome staining was carried out for determination of myocardial fibrosis according to standard procedures (Puente et al., 2014; Deng et al., 2016) while haematoxylin-eosin (HE) staining was performed to observe the arrangement and morphology of cardiomyocytes (Wu et al., 2017).

Similar to a previous study (Bing et al., 2020), the equation for the pulsatile flow velocity profile across the lumen, u(r,t), is given as:

where r is the radial coordinate, R is the radius of artery, Λ² = i³α², α=R⁢ω⁢ρμ, q_measured(t) = Q(ω)e^iωt, ω is the angular frequency after Fourier transformation, J₀is a Bessel function of zero order and first kind, and J₁ is a Bessel function of first order and first kind. Accordingly, wall shear stress (WSS), τ(R,t), and oscillatory shear index (OSI) for pulsatile blood flow can be written as:

The viscosity (μ) and density (ρ) were assumed to be 4.0 cp and 1.06 g/cm³, respectively. Moreover, relative residence time (RRT) reflects the residence time of flow particles near the wall and is recommended as a single metric of low oscillating shear stress, which is expressed as follows:

The experimental measurements were repeated three times and averaged per animal. All parameters were represented as mean ± S.E.M. by averaging over all animals in each group. A two-way ANOVA (SigmaStat 3.5) was used to detect the statistical difference of morphometric and hemodynamic parameters between sham and MI groups and between inhalation of zinc particle and no inhalation groups, where P < 0.05 was indicative of a significant difference between the two populations.

Myocardial infarction resulted in cardiac impairments, such as LV wall thinning, LV cavity dilation, remarkable decrease of EF, FS, dp/dt Max, and −dp/dt Min, and increased LVEDP and Tau as well as decreased systolic and diastolic blood pressures for 4 weeks after LAD ligation. The short-term inhalation of ultrafine zinc particles slowed down cardiac impairments caused by the myocardial infarction. The role of zinc as a modulator of cardiac changes has been recognized, which could affect homeostasis, oxidative stress, immune function, apoptosis, and ageing (Chasapis et al., 2012; Andrea et al., 2018). Zinc, a vital antioxidative element, can protect myocyte membrane against unsaturated lipids and inflammatory cytokines (Hennig et al., 1996) and thus alleviate the infarction-induced heart failure (Shokrzadeh et al., 2009) albeit there was much debate about the role of zinc concentrations in different types of cardiomyopathy (Marin and Rodriguez-Martinez, 1995; Kosar et al., 2006). A meta-analysis indicated a significant association between zinc deficiency and MI (Liu et al., 2015). The short-term inhalation of ultrafine zinc particles alleviated myocardial dysfunctions and slowed down conversion of MI to heart failure in MI rats, which agreed with previous studies that EF increased with Zn content (Oster et al., 1993) and Zn supplementation to a cardioplegic solution reversed the loss of systolic function (Powell et al., 1995).

Myocardial fibrosis makes a critical contribution to the LV remodeling in MI-induced HFrEF. Ultrafine zinc particles entered the systemic circulation through the respiratory system and suppressed tissue collagen deposition by inhibiting proline hydroxylase activity (Mezey et al., 1976; Mann et al., 1979; Camps et al., 1992), and regulated metabolism of fiber collagen (Wang et al., 2005). Wang et al. (2005) have shown that myocardial fibrosis was related to metallothionein (MT) regulation of zinc homeostasis and zinc supplementation prevented the fibrotic process in the MT-KO mice. Inhaling ultrafine zinc particles reduced the degree of myocardial fibrosis and hence slowed down conversion of MI to heart failure in MI rats.

The MI group had a significant decrease of systolic and diastolic blood pressures. The short-term inhalation of ultrafine zinc particles stimulated peripheral arteries and arterioles to elevate systolic and diastolic blood pressures in the MI-Zn group, which can be another factor for Zn protection against the MI-induced heart failure. On the other hand, we have shown a significant increase of systolic and diastolic blood pressures in rats of HFpEF (Bing et al., 2020). The short-term inhalation of ultrafine zinc particles further increased the systolic and diastolic blood pressures and hence deteriorated cardiac and hemodynamic environment in rats with HFpEF.

Myocardial strain and strain rate characterize the extent of cardiac deformation (Niu et al., 2020). In the STE analysis, the longitudinal strain refers to the shortening of myocardium fibers from the base to the apex, the circumferential strain represents the circumferential shortening observed in the short-axis view (Saito et al., 2009), and the radial strain shows the myocardial shortening moving from the LV center to the periphery (Heimdal, 1998). Accordingly, the strain rates refer to the relaxation of myocardium fibers (Niu et al., 2020). The MI-induced decrease of longitudinal, circumferential and radial strains and strain rates denoted the significant impairment of systolic and diastolic functions, respectively. The short-term inhalation of ultrafine zinc particles slowed down the progression of systolic and diastolic dysfunctions. In mammalian myocytes, it is known that Zn²⁺ is of importance to regulate the excitation-contraction coupling (Turan et al., 1997; Tuncay et al., 2011). Woodier and co-workers showed that cytosolic Zn²⁺ acted as a high affinity activator of RyR2 and modulated the frequency and amplitude of Ca²⁺ waves in myocytes in a concentration-dependent manner (Woodier et al., 2015). Defective Zn²⁺ handling enhanced the impaired contractility in myocytes (Little et al., 2010). Inhaling ultrafine zinc particles significantly increased the serum zinc concentration. Zinc transporter Zip14 can transfer the elevated extracellular zinc into cardiomyocytes (Taylor et al., 2005; Kim et al., 2017; Yusuf et al., 2018) to alleviate systolic and diastolic dysfunctions in MI rats.

We investigated the hemodynamic changes in the carotid artery of the four groups. The Womersley analysis showed ∼39% reduction of TAWSS in the MI group as compared with the shams. TAWSS in the Sham-Zn group was ∼28% higher than that in the Sham group while the value in the MI-Zn group was ∼44% higher than that in the MI group. This was caused by the increased CO and SV after short-term inhalation of ultrafine zinc particles. Moreover, OSI is high in the MI group despite negligible values in other groups. RRT in the MI group was ∼60% higher than the shams while the value in the MI-Zn group was ∼27% lower than that in MI group. The short-term inhalation of ultrafine zinc particles alleviated the hemodynamic environment in the carotid artery, such as the increased TAWSS, the decreased OSI, and the reduced RRT. These abnormal parameters are known to result in endothelial dysfunction, monocyte deposition, SMC proliferation, microemboli formation, and so on (Huang et al., 2016; Han et al., 2018; Huang X. et al., 2018; Fan et al., 2019). It was reported that MI-induced zinc deficiency aggravates pro-inflammatory and impairs anti-inflammatory responses in vascular endothelial cells though activation of NF-κB and inhibition of PPAR pathways (Connell et al., 1997; Li and Karin, 1999; Chung et al., 2000; Delerive et al., 2000; Hihi et al., 2002; Griendling and FitzGerald, 2003). Zinc supplementation could function as the anti-inflammatory (Jarosz et al., 2017), preventing endothelial cell dysfunction, and subsequent cardiovascular diseases (Shen, 2008). Hence, the short-term inhalation of ultrafine zinc particles may protect peripheral arteries from the hemodynamic impairments, which still required more investigations.

We only measured the serum zinc levels, but not the concentration in myocardial tissues. The proportion of serum zinc entering cardiomyocyte via zinc transporter Zip14 is required to be investigated in the following studies. A further limitation is the lack of inclusion of a control particle, for example, an inert particle of the same size which is identified harmless to body. This is essential to demonstrate that the effects are down to zinc per se, rather than simply particulate matter. The present study only considered the effects of short-term inhalation on cardiac function and peripheral cardiovascular hemodynamics. The long-term inhalation of ultrafine zinc particles should be included in the following studies. Moreover, histological analysis and cellular and molecular mechanisms are still required for investigations.